Methods of preparing personalized blood vessels

ABSTRACT

The present disclosure relates to methods of preparing personalized blood vessels, useful for transplantation with improved host compatibility and reduced susceptibility to thrombosis. Also provided are personalized blood vessels produced by the methods and use thereof in surgery.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/714,200, filed Aug. 3, 2018.

FIELD OF DISCLOSURE

The present disclosure relates to personalized blood vessels and methodsof preparing personalized blood vessels. In various embodiments thepersonalized blood vessels produced by the methods contemplated hereinmay be useful in transplantation with improved host compatibility,tissue integration and reduced susceptibility to thrombosis.

BACKGROUND

Vascular diseases constitute a serious and constantly growing healthissue on a global scale. The options for replacement of damaged bloodvessels are limited today. Autologous transplantation requires suitablevessel in the patient and leads to donor site morbidity. Syntheticvessel replacements are available, but these lack biological integrationin the host tissue and long-term patency. There is a need forfully-biological patient-individualized grafts.

International publication number WO/2013/136184 discloses “methods forrecellurization of blood vessels;” for example “for producing anallogeneic vein, wherein a donor vein is decellularized and thenrecellularized using whole blood or bone marrow stem cells.”

International publication number WO/2015/181245 discloses “methods forrecellularization of valves in valve-bearing veins;” for example “forproducing an allogeneic venous valve, wherein a donor valve-bearing veinis decellularized and then recellularized using whole blood or bonemarrow stem cells.”

SUMMARY OF THE DISCLOSURE

One aspect of the present disclosure relates to a method of preparing apersonalized blood vessel comprising, contacting a surface of anacellular tubular scaffold with an undiluted whole blood sample from asubject in need of the personalized blood vessel, wherein the contactingis performed for more than 2 days.

Another aspect of the present disclosure relates to a method ofpreparing a personalized blood vessel, comprising contacting a surfaceof an acellular tubular scaffold with a suspension comprising a wholeblood sample from a subject in need of the personalize blood vessel,wherein the whole blood sample is diluted in a physiological solution.In some embodiments, the physiological solution maintains colloidoncotic pressure of the extracellular environment, buffers the pH in aCO₂ independent matter, and/or provides a cell protective or antioxidanteffect.

In some embodiments, a population of cells present in the whole bloodsample populates the scaffold.

In some embodiments, the whole blood sample comprises one or morenon-cellular factors, wherein one or more non-cellular factors of thewhole blood populate the scaffold, and wherein the one or morenon-cellular factors promote cellularization of the acellular tubularscaffold and host compatibility of the vessel upon grafting.

In some embodiments, the undiluted whole blood sample or the suspensioncomprising the whole blood sample further comprises an anti-thromboticfactor.

In some embodiments, the anti-thrombotic factor comprises ananticoagulant agent.

In some embodiments, the anticoagulant agent comprises heparin or adextran.

In some embodiments, the heparin is present in the undiluted whole bloodsample or the suspension comprising the whole blood sample at aconcentration from about 0.5 IU/mL to about 150 IU/mL at the beginningof contacting the surface of the acellular tubular scaffold.

In some embodiments, the heparin is present in the undiluted whole bloodsample or the suspension comprising the whole blood sample at aconcentration of about 6.7 IU/mL at the beginning of contacting thesurface of the acellular tubular scaffold.

In some embodiments, the dextran is dextran-40.

In some embodiments, the dextran is present in the undiluted whole bloodsample or the suspension comprising the whole blood sample at aconcentration from about 1 g/L to about 55 g/L at the beginning ofcontacting the surface of the acellular tubular scaffold.

In some embodiments, the anti-thrombotic agent comprises ascorbic acid.

In some embodiments, the ascorbic acid is present in the undiluted wholeblood sample or the suspension comprising the whole blood sample at aconcentration from about 0.2 μg/mL to about 200 μg/mL at the beginningof contacting the surface of the acellular tubular scaffold.

In some embodiments, the concentration of ascorbic acid is present inthe undiluted whole blood sample or the suspension comprising the wholeblood sample at a concentration of about 5 μg/mL at the beginning ofcontacting the surface of the acellular tubular scaffold.

In some embodiments, the anti-thrombotic factor comprisesacetylsalicylic acid.

In some embodiments, the acetylsalicylic acid is present in theundiluted whole blood sample or the suspension comprising the wholeblood sample at a concentration of from about 0.2 μg/mL to about 200μg/mL at the beginning of contacting the surface of the acellulartubular scaffold.

In some embodiments, the acetylsalicylic acid is present in theundiluted whole blood sample or the suspension comprising the wholeblood sample at a concentration of about 5 μg/mL at the beginning ofcontacting the surface of the acellular tubular scaffold.

In some embodiments, the whole blood sample or the suspension comprisingthe whole blood sample further comprises equal to or more than thepopulation average physiological level of a growth factor selected fromthe group consisting of: granulocyte macrophage-colony stimulatingfactor (GM-CSF), interleukin (IL)-3, IL-4, neutrophin (NT)-6,pleiotrophin (HB-GAM), midkine (MK), interferon inducible protein-10(IP-10), platelet factor (PF)-4, monocyte chemotactic protein-1 (MCP-1),RANTES (CCL-5, chemokine (C-C motif) ligand 5), IL-8, IGFs, fibroblastgrowth factor (FGF)-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8,FGF-9, transforming growth factor (TGF)-β, VEGF, platelet-derived growthfactor (PDGF)-A, PDGF-B, HB-EGF, hepatocyte growth factor (HGF), tumornecrosis factor (TNF)-α, insulin-like growth factor (IGF)-1, and anycombination(s) thereof.

In some embodiments, the growth factor is a fibroblast growth factor(FGF)-2.

In some embodiments, the FGF-2 is human FGF-2.

In some embodiments, the FGF-2 is present in the undiluted whole bloodsample or the suspension comprising the whole blood sample at aconcentration from about 0.5 ng/mL to about 200 ng/mL at the beginningof contacting the surface of the acellular tubular scaffold.

In some embodiments, the concentration of the FGF-2 is present in theundiluted whole blood sample or the suspension comprising the wholeblood sample at a concentration of about 10 ng/mL at the beginning ofcontacting the surface of the acellular tubular scaffold.

In some embodiments, the growth factor is a vascular endothelial growthfactor (VEGF).

In some embodiments, the VEGF is human VEGF.

In some embodiments, the VEGF is present in the undiluted whole bloodsample or the suspension comprising the whole blood sample at aconcentration from about 4 ng/mL to about 800 ng/mL at the beginning ofcontacting the surface of the acellular tubular scaffold.

In some embodiments, the VEGF is present in the undiluted whole bloodsample or the suspension comprising the whole blood sample at aconcentration of about 80 ng/mL at the beginning of contacting thesurface of the acellular tubular scaffold.

In some embodiments, the whole blood sample or the suspension comprisingthe whole blood sample further comprises human serum albumin, whereinthe concentration of human serum albumin at the beginning of contactingthe surface of the acellular tubular scaffold is about 55 g/L to about105 g/L.

In some embodiments, the physiological solution comprises an inorganicsalt, and a buffer system.

In some embodiments, the buffer system comprises a CO2-independentbuffer system

In some embodiments, the CO2-independent buffer system comprises aphosphate buffer system.

In some embodiments, the physiological solution exhibits an osmoticpressure substantially similar to whole blood, which preferably is fromabout 270 mOsm/kg H₂O to about 310 mOsm/kg H₂O.

In some embodiments, the physiological solution further comprises anoncotic factor and/or a nutrient.

In some embodiments, the oncotic factor comprises serum albumin.

In some embodiments, the concentration of human serum albumin VEGF ispresent in the undiluted whole blood sample or the suspension comprisingthe whole blood sample at a concentration from about 55 g/L to about 105g/L at the beginning of contacting the surface of the acellular tubularscaffold.

In some embodiments, the nutrient comprises one or more of a sugar, anamino acid, or a vitamin.

In some embodiments, the sugar is D-glucose.

In some embodiments, the physiological solution comprises a growthfactor, and antithrombotic factor, a nutrient, and an oncotic factor;wherein the growth factor comprises human FGF-2 and human VEGF; whereinthe anti-thrombotic factor comprises one or more of acetylsalicylicacid, heparin or dextran; and wherein the nutrient comprises D-glucose.

In some embodiments, the method does not require contacting theacellular tubular scaffold with a cell culture medium after contactingwith whole blood for preparing the personalized blood vessel.

In some embodiments, contacting the surface of the acellular tubularscaffold is for more than 3 days, more than 4 days, more than 5 days,more than 6 days, more than 7 days, for 2 to 21 days, for 3 to 21 days,for 4 to 21 days, for 5 to 21 days, for 6 to 21 days, for 7 to 21 days,or for 7-9 days.

In some embodiments, wherein the acellular tubular scaffold is contactedwith the suspension comprising the whole blood sample, and the methodfurther comprises monitoring a plurality of environmental parametersand/or a concentration of a nutrient during the preparation of thepersonalized blood vessel.

In some embodiments, the methods of the present disclosure furthercomprise adjusting the plurality of environmental parameters.

In some embodiments, the plurality of environmental parameters comprisestemperature, pH, oxygen, and/or CO₂.

In some embodiments, the acellular tubular scaffold is contacted withthe suspension comprising the whole blood sample, and the method furthercomprises monitoring a concentration of a nutrient in the suspension.

In some embodiments, the methods of the present disclosure furthercomprise continuously or regularly adjusting the concentration of thenutrient.

In some embodiments, the nutrient is D-glucose, and wherein theD-Glucose concentration is adjusted continuously or regularly tomaintain the D-glucose concentration at about 3 to about 11 mmol/L.

In some embodiments, the concentration of D-glucose in the suspension ismonitored by measuring the concentration of D-glucose in a samplecollected from the suspension.

In some embodiments, the concentration of D-glucose in the suspension ismeasured once every day.

In some embodiments, the concentration of D-glucose in the suspension ismonitored by measuring the concentration of D-glucose using a sensorthat is in contact with the suspension.

In some embodiments, the sensor is continuously in contact with thesuspension during the contacting.

In some embodiments, D-glucose is added when the measured concentrationof D-glucose in the suspension is below 4 mmol/L.

In some embodiments, D-glucose is added to reach a final concentrationof 10 mmol/L of D-glucose in the suspension.

In some embodiments, the surface of the acellular tubular scaffold isthe inner surface of the acellular tubular scaffold.

In some embodiments, the whole blood comprises peripheral blood orumbilical cord blood.

In some embodiments, the method further comprises monitoring aconcentration of human serum albumin, wherein the concentration of humanserum albumin is about 55 g/L to about 105 g/L at the beginning ofcontacting the surface of the acellular tubular scaffold.

In some embodiments, wherein contacting the acellular tubular scaffoldresults in proliferation and/or differentiation of a plurality ofprogenitor cells to a plurality of endothelial cells.

In some embodiments, the plurality endothelial cells expressVE-cadherin, AcLDL, vWF, and/or CD31.

In some embodiments, the acellular tubular scaffold is a decellularizedblood vessel.

In some embodiments, the acellular tubular scaffold is continuouslyperfused with the suspension or whole blood.

In some embodiments, the acellular tubular scaffold is perfused at aspeed of about 0.1 mL to about 50 mL per minute.

In some embodiments, the acellular tubular scaffold is perfused at aspeed of about 2 mL per minute.

In some embodiments, the scaffold is perfused with a closedrecirculation of the suspension or whole blood.

In some embodiments, the continuous contacting is perfusion conductedusing a bioreactor.

In some embodiments, the contacting is conducted in vitro.

In some embodiments, the contacting is conducted at about 8° C. to about40° C.

In some embodiments, wherein the contacting is conducted at about 20° C.to about 25° C.

In some embodiments, the personalized blood vessel is a vein.

In some embodiments, the vein is a femoral vein.

In some embodiments, the methods of the present disclosure furthercomprise assessing the venous valve function of the personalized bloodvessel using a valve competence test.

Another aspect of the present disclosure relates to a personalized bloodvessel prepared by the method of any one of the methods of preparing apersonalized blood vessel disclosed herein.

In some embodiments, the personalized blood vessel has been implantedinto a subject by surgery.

Another aspect of the present disclosure relates to a method of surgerycomprising implanting the personalized blood vessel disclosed hereininto a subject in need thereof.

Another aspect of the present disclosure relates to a personalized bloodvessel for use in a method of surgery, wherein the method comprisesimplanting the personalized blood vessel of disclosed herein into asubject in need thereof.

In some embodiments, the subject is human.

Another aspect of the present disclosure relates to a bioreactor forpreparing a personalized blood vessel, the bioreactor comprising aperistaltic pump, a container comprising a sampling port, a firstconnector, and a second connector, wherein the first and secondconnectors are directly or indirectly connected to the container,wherein each connector is connected to an end of a tubular scaffold forpreparing a personalized blood vessel prepared by the methods disclosedherein, wherein when the first and second connectors are connected tothe two ends of a tubular scaffold, the peristaltic pump mediates thecirculation of a suspension or solution in a closed circuit.

In some embodiments, the first and second connectors are Luerconnectors.

In some embodiments, the sampling port is an injection port.

In some embodiments, the bioreactor further comprises an injection port.

In some embodiments, the injection port is connected to a reservoir ofD-glucose.

In some embodiments, the first container is directly connected to thecontainer by a tube, and/or the second container is directly connectedto the container by a tube.

In some embodiments, the bioreactor comprises one or more sample ports.

In some embodiments, the bioreactor further comprises one or moresensors for measuring glucose level.

In some embodiments, the bioreactor further comprises a pH adjustingmodule.

In some embodiments, the bioreactor further comprises a CO₂ adjustingmodule.

Another aspect of the present disclosure relates to a method ofpreparing a personalized blood vessel, the method comprising contactinga surface of an acellular tubular scaffold with a component contained inthe whole blood, which is enriched or selected prior to use incontacting the surface.

In some embodiments, the component is selected from thrombocytes,nucleated cells, proteins, growth factors, signaling factors,immunoglobulins, and any combinations thereof.

In some embodiments, the component is enriched by centrifugation,gradient centrifugation; or selected by selective adhesion, filtration,or sorting.

In some embodiments, the surface is an inner surface of the acellulartubular scaffold.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are angiograms of a sham operated vena cava (FIG. 1A) and atransplanted P-TEV (FIG. 1B). Metal clips are indicated by arrow headsto localize the anastomoses.

FIGS. 2A-2B are a series of images showing Hematoxylin and Eosin (H&E)staining and 4′,6-diamidino-2-phenylindole (DAPI) staining of nativevena cava (“Native,” FIG. 2A) and P-TEV transplants (“P-TEV,” FIG. 2B)from adjacent sections 3 days, 2 weeks, 17 days, and 4-5 weekspost-surgery.

FIGS. 3A-3D depict images showing vena cava cellularization two weekspost surgery. FIGS. 3A-3D are a series of immunohistograms showing DAPIstaining and CD31 immunostaining of vena cava two weeks post-surgery.FIG. 3A shows native vena cava proximal to the anastomoses.

FIGS. 3B-3D show proximal (FIG. 3B), center (FIG. 3C), and distal (FIG.3D) parts of the P-TEV. Arrow heads indicate CD31-positive cells.

FIGS. 4A-4D depict images showing Vena cava cellularization four weekspost surgery. FIGS. 4A-4D are a series of immunohistograms showing DAPIstaining and CD31 immunostaining of vena cava 4-5 weeks post-surgery.FIG. 4A shows native vena cava proximal to the anastomoses. FIGS. 4B-4Dshow proximal (FIG. 4B), center (FIG. 4C), and distal (FIG. 4D) parts ofthe P-TEV. Arrow heads indicate CD31-positive cells.

FIGS. 5A-5B depict images showing luminal cell morphology four weekspost surgery. FIGS. 5A-5B is a series of images at 40× magnificationshowing H&E staining of native vena cava (FIG. 5A) and P-TEV transplant(FIG. 5B) 4-5 weeks post-surgery. Arrows indicate endothelial cells inthe native tissue and cells with plated endothelial cell-like morphologyin the P-TEV transplant.

DETAILED DESCRIPTION OF THE DISCLOSURE

The instant disclosure is based, inter alia, upon the discovery thatcells and non-cellular factors in the blood were able to conditionacellular tubular scaffolds (e.g., a decellularized blood vessel or abioprinted tubular scaffold) to produce personalized blood vessels. Theinstant disclosure further provides, inter alia, contacting acellulartubular scaffolds with either undiluted whole blood samples or asuspension comprising a whole blood sample diluted in a physiologicalsolution in an in vitro process produces personalized blood vessels withimproved host compatibility and reduced susceptibility to thrombosis.

Accordingly, one aspect of the present disclosure relates to a method ofpreparing a personalized blood vessel comprising, contacting a surfaceof an acellular tubular scaffold with an undiluted whole blood samplefrom a subject in need of the personalized blood vessel, wherein thecontacting is performed for more than 2 days.

Another aspect of the present disclosure relates to a method ofpreparing a personalized blood vessel, comprising contacting a surfaceof an acellular tubular scaffold with a suspension comprising a wholeblood sample from a subject in need of the personalize blood vessel,wherein the whole blood sample is diluted in a physiological solution.In some embodiments, the surface of the acellular tubular scaffold isthe inner surface of the acellular tubular scaffold.

As used herein, the term “personalized blood vessel” refers to a tubularstructure capable of carrying blood through a tissue or organ that isnot a native blood vessel. The tubular structure can be prepared bydecellularizing a native blood vessel from a donor, thereby obtaining anacellular scaffold, and reconditioned/recellularizing the scaffold usingcells from another individual. An acellular scaffold can also beobtained by assembling a biocompatible material in vitro (e.g.,bioprinting or polymer self-assembly). As used herein, the terms“recondition,” “reconditioning,” and “reconditioned” are used todescribe the modification of the acellular scaffold by components of therecellularization/recondition (RC) solution containing whole blood. Asused herein, the terms “recellularize,” “recellularizing,” and“recellularization” are used in either context, and do not require thatthe initial starting material has cells attached. A personalized bloodvessel can be a personalized vein, artery, or capillary. In certainembodiments, a personalized blood vessel is useful to replace a nativeblood vessel.

Preparation of the Acellular Tubular Scaffold from Donor

The method of preparing a personalized blood vessel as disclosed hereinemploys an acellular tubular scaffold. As used herein, the term“acellular tubular scaffold” refers to a tubular scaffold that issubstantially cell-free. An acellular tubular scaffold can be preparedby decellularizing a native blood vessel from a donor. The donor can bea human or an animal from a suitable species. An acellular scaffold canalso be obtained by assembling a biocompatible material in vitro (e.g.,bioprinting or polymer self-assembly).

The acellular tubular scaffold can be prepared by any method known inthe art, for example, decellularization of a native blood vessel. Thenative blood vessel can be of human origin or can be obtained fromsuitable animal species. A tubular structure can be prepared bytissue-culture methods from biocompatible scaffold and cells in vitroand can serve as the starting material for the preparation of anacellular scaffold. In certain embodiments, the acellular tubularscaffold is obtained by a method of decellularization disclosed herein.In certain embodiments, the method of preparing a personalized bloodvessel disclosed herein further comprises decellularizing a native bloodvessel, thereby obtaining an acellular tubular scaffold.

As used herein, the term “decellularize,” “decellularized,”“decellularizing,” or “decellularization” refers to the process ofremoving cells from a blood vessel (including any venous valve in theblood vessel). Effective decellularization is dictated by factors suchas tissue density and organization, geometric and biologic propertiesdesired for the end product, and the targeted clinical application.Decellularization of blood vessels with preservation of the ECMintegrity and bioactivity can be optimized by those skilled in the art,e.g., by choosing specific agents and techniques during processing.

Successful decellularization is defined as the absence cells, such asendothelial cells, smooth muscle cells, and nuclei in histologicsections using standard histological staining procedures. While mostcell removal agents and methods may alter ECM composition and cause somedegree of ultrastructure disruption, minimization of these undesirableeffects is desired. Methods of decellularization are known in the art,e.g., in U.S. Patent Publication No. 2017/0071738.

Accordingly, in certain embodiments, the decellularization processremoves all nuclei, as detected by a method known in the art (e.g., DAPIstaining, DNA quantification). In certain embodiments, thedecellularization process reduces or removes HLA class-I antigens andHLA class-II antigens, as detected by a method known in the art (e.g.,immuohistochemical assays for HLA class-I and class-II antigens).

In certain embodiments, the ECM components are substantially retained inthe decellularization process. During and following the process ofdecellularization, the morphology and architecture of the ECM can beexamined visually and/or histologically to verify that thedecellularization process has not compromised the three-dimensionalstructure and bioactivity of the ECM scaffold. Histological analysis bystaining (e.g., H&E staining, Masson's Trichrome staining, orVerhoeff-Van Gieson staining) may be useful to visualize decellularizedblood vessel architecture and preservation of ECM components, such ascollagen I, collagen IV, laminin and fibronectin. Other methods known inthe art may be used for determining the preservation of ECM components,such as glycosaminoglycans and collagen.

One or more cellular disruption solutions may be used to decellularize ablood vessel. A cellular disruption solution generally includes at leastone detergent, such as SDS, PEG, or Triton X. A particularly preferreddetergent is Triton X (e.g., Triton X-100). A cellular disruptionsolution may comprise an organic solvent, e.g., Tri-n-Butyl Phosphate(TNBP). A cellular disruption solution can also have a substantiallylower osmotic pressure than blood, thereby facilitating cell lysis.Alternatively, a cellular disruption solution can be isotonic. Cellulardisruption solution also can include enzymes such as, withoutlimitation, one or more nucleases (e.g., DNases or RNases) andproteinases (e.g., collagenases, dispases, or trypsin). A cellulardisruption solution that comprises DNase I may also include calciumchloride and magnesium chloride (A12858, Life Technologies) to activatethe enzyme. In certain embodiments, a cellular disruption solutionfurther comprises one or more protease inhibitors (e.g., phenyl methylsulfonyl fluoride (PMSF), collagenase inhibitors). In certainembodiments, a cellular disruption solution may further include anantibiotic (e.g., penicillin, streptomycin, or amphotericin) orethylenediaminetetraacetic acid (EDTA).

In certain embodiments, perfusion methods may be used to treat the bloodvessel (e.g., valve-bearing vein) with cellular disruption solutions fordecellularization of the blood vessel. Alternating the direction ofperfusion (e.g., antegrade and retrograde) can help to effectivelydecellularize a valve-bearing veins. Decellularization as describedherein essentially removes the cells lining the valve-bearing veins fromthe inside out, resulting in very little damage to the ECM. Dependingupon the size and weight of the blood vessel and the particulardetergent(s) and concentration of detergent(s) in the cellulardisruption solution, a valve-bearing vein is generally perfused between4 and 48 hours; or between 8 and 24 hours with cellular disruptionmedium. Including washes, a blood vessel may be perfused for up to about48 hours or 24 hours for Triton X, about 8 hours for TNBP, about 16hours for DNase, about 1 hour total washes and about 48 hours finalwash. In some embodiments the aforementioned profusion procedure isrepeated for 1-14 cycles; 1-5 cycles; 2-4 cycles; 1-3 cycles; or 2cycles; or 3 cycles; or 4 cycles; or 5 cycles; or 6 cycles. In certainembodiments, the decellularization is performed by perfusion with theaforementioned reagents continuously for 100 hours, 110 hours, 120hours, 130 hours, 140 hours, 150 hours, 160 hours, 170 hours, 180 hours,190 hours, 200 hours, 210 hours, 220 hours, 230 hours, 240 hours, 250hours, 260 hours, 270 hours, 280 hours, 290 hours, 300 hours, 310 hours,320 hours, 330 hours, 340 hours, 350 hours, or more. Perfusion generallyis adjusted to physiologic conditions including pulsatile flow, rate andpressure. Perfusion decellularization as described herein can in someembodiments be compared to immersion decellularization as described, forexample, in U.S. Pat. Nos. 6,753,181 and 6,376,244. In certainembodiments, the decellularization is conducted according to the methodof decellularizing a blood vessel as provided in Example 1 infra.

In certain embodiments, the acellular tubular scaffold is a synthesizedand/or assembled scaffold, e.g., a scaffold obtained by assembling abiocompatible material in vitro. Methods of preparing such scaffold,e.g., bioprinting or polymer self-assembly, are known in the art, andnon-limiting examples are provided in U.S. Patent Publication Nos.2017/0304503 and 2016/0354194.

Accordingly, in certain embodiments, the method disclosed herein furthercomprises a step of preparing a bioprinted tubular blood vessel scaffoldusing a bioprinted polymeric scaffold. The bioprinted blood vesselscaffold is prepared on a polymer (natural or synthetic), which may bein a gel form, sponge form, foam form, patch form, or asemi-liquid/fluid form. In certain embodiments, a bioprinted scaffold isperfused with whole blood or whole blood diluted in a solution, e.g., asuspension, which includes whole blood.

In certain embodiments, the ECM components may be added to the polymerin a powder form. In the present disclosure the powder for preparing thecomposition of the present disclosure is prepared by treating a tissuewith a chemical, freeze-drying the chemical treated tissue, andhomogenization of freeze-dried tissue. In certain embodiments, thepowder is filamentous.

In certain embodiments, bioprinted polymeric scaffold may includegelatin and fibrin. The gelatin and fibrin may form an interpenetratingpolymer network that mimics natural ECM and may be optimized for cellattachment, bioprinting, transparency, and biocompatibility.

In certain embodiments, components of the ECM are or may be isolatedand/or purified from a tissue of a mammal (e.g., a pig, a cow, a lamb, agoat, a sheep, a chimpanzee, a monkey, a human, or other primate) orgenerated using recombinant DNA technology involving gene or genefragments of a mammal (e.g., a pig, a cow, a lamb, a goat, a sheep, achimpanzee, a monkey, a human, or other primate) encoding the respectiveECM component (e.g., collagens, elastins, laminins, glycosaminoglycans,proteoglycans, antimicrobials, chemoattractants, cytokines, and growthfactors) and expressed in from a suitable expression system (prokaryoticor eukaryotic (e.g., yeast, insect, or mammalian cells)), andsubsequently isolated and/or purified by suitable method in the art. Incertain embodiments, one or more of the ECM components may be added tothe polymeric scaffold for preparing a bioprinted polymeric scaffold,and then the scaffold is perfused with whole blood or whole blooddiluted in a solution, e.g., a suspension, which includes whole blood,for preparing a personalized blood vessel.

In some embodiments, the tubular scaffold may be prepared by methodssuch as 3D-printing of different materials, self-assembly, chemical orbiochemical manufacturing. In some embodiments, the tubular scaffold ismanufactured from biological and non-biological starting material.

Patient Whole Blood

In some embodiments, a population of cells present in the whole bloodsample populates the scaffold. The patient whole blood may be usedundiluted or diluted with a physiological perfusion solution such asdescribed in the examples 4-5 or other organ preservative solutions suchas for example Perfadex™, STEEN™, Euro Collins solution, University ofWisconsin cold storage solution or Celsior® solution.

In certain embodiments, the whole blood comprises peripheral blood(e.g., peripheral venous blood). In certain embodiments, the whole bloodis peripheral blood (e.g., peripheral venous blood). In certainembodiments, the whole blood comprises umbilical cord blood. In certainembodiments, the whole blood is umbilical cord blood.

The terms “patient” and “subject” are used interchangeably in thisdisclosure and refer to a human or non-human animal (e.g., a mammal).Non-limiting examples include humans, bovines, rats, mice, dogs, cats,monkeys, goat, sheep, cows, and deer. In some embodiments, the subjectis a human.

Undiluted Patient Whole Blood

One aspect of the present disclosure relates to a method of preparing apersonalized blood vessel comprising, contacting a surface of anacellular tubular scaffold with an undiluted whole blood sample from asubject in need of the personalized blood vessel, wherein the contactingis performed for more than 2 days.

In certain embodiments, the cells and/or progenitor cells present in theblood populate the scaffold. In some embodiments, a population of cellspresent in the whole blood sample populates the scaffold. The term“whole blood” refers to blood drawn from a body, organ, or tissue, fromwhich none of the components has been removed.

The skilled person would appreciate that during the collection of wholeblood, a small volume of an anti-thrombotic agent may be added during orimmediately after the blood sample is drawn from a subject. Where thevolume of the anti-thrombotic agent does not exceed about 5% or at leastnot 10% of the volume of the blood with which the anti-thrombotic agentis mixed, the whole blood thereby prepared is considered undiluted.Similarly, in certain embodiments, the whole blood may be supplementedwith one or more other agents (e.g., anti-thrombotic agents, nutrients,diluent, preservatives, and CO₂-independent buffer system), wherein thetotal volume of the added substance(s)—not including any anti-thromboticagent initially introduced at the time of collection—does not exceedabout 5% or at least not about 10% of the volume of the whole blood, thesupplemented whole blood is considered undiluted.

In certain embodiments, the method does not require contacting theacellular tubular scaffold with a cell culture medium for preparing thepersonalized blood vessel. In certain embodiments, the whole blood isused in the method disclosed herein within about 1 hour, about 2 hours,about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 24hours, about 36 hours, about 2 days, about 3 days, or about 1 week afterthe collection of the whole blood. In certain embodiments, the wholeblood is stored at about 2° C. to about 8° C. before use in the method.

Diluted Patient Whole Blood

In another aspect, the present invention relates to contacting theacellular tubular scaffold with diluted patient whole blood.

The patient whole blood is considered diluted if the whole blood drawnfrom the patient is diluted with any type of solution, buffer, oraqueous liquid at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. Insome embodiments, the whole blood sample is diluted 1:1, i.e. 1 volumeof whole blood is mixed with 1 volume diluent. In some embodiment, thewhole blood sample may be diluted 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, or 1:10.

In certain embodiments, the whole blood is diluted in a physiologicalsolution. In certain embodiments, the diluent is a physiologicalsolution containing additional oncotic and other factors as described inthe examples x-y or other organ preservative solutions such as forexample Perfadex™ (see Ex. 5 below), STEEN™ (non-toxic physiologicalsalt solution comprising human serum albumin and dextran 40, or asolution as described in U.S. Pat. No. 8,980,541) Euro Collins solution(see Ex. 5 below), University of Wisconsin cold storage solution (seeEx. 5 below) or Celsior® solution (see Ex. 5 below). In certainembodiments, the physiological solution is STEEN™ solution.

In some embodiments, the oncotic factors contemplated to be use toregulate the osmotic pressure of the physiological solution compriseserum albumin, globulin, or fibrinogen. In some embodiments, the oncoticfactor comprises serum albumin. In some embodiments, gelatin may be usdto regulate the oncotic pressure of the solution.

A physiological solution may further comprise one or more nutrients(e.g., amino acid, monosaccharide (e.g., D-glucose), vitamin, inorganicion, trace element, and/or salt) and/or growth factors, e.g., atphysiological concentrations, to support survival, proliferation, and/ordifferentiation of cells. In some embodiments, the nutrient comprisesone or more of a sugar, an amino acid, or a vitamin. In someembodiments, the sugar is D-glucose.

In some embodiments, the physiological solution comprises an inorganicsalt, and a buffer system. In some embodiments, the buffer systemcomprises a CO₂-independent buffer system. In some embodiments, theCO₂-independent buffer system comprises a phosphate buffer system. Thephysiological solution may contain any of the further agents to be addedto the whole blood or diluted whole blood as described below.

Further Agents Added to Whole Blood or Diluted Whole Blood Samples

In some embodiments, further agents described herein are added to thewhole blood sample or to the suspension of whole blood or the dilutedwhole blood sample before or during contacting the acellular tubularscaffold with the whole blood. As explained above, as long as theaddition of the factors described herein does not dilute the whole bloodwith more than 5 or at least not more than 10%, the whole blood sampleis still considered undiluted. If the whole blood sample is diluted morethan 15% with the factors described herein, then the whole blood sampleis diluted.

In certain embodiments, the suspension with whole blood or diluted wholeblood or the undiluted whole blood further comprises an anti-thromboticagent. In certain embodiments, an anti-thrombotic agent is or has beenintroduced to the whole blood before contacting the acellular tubularscaffold. In certain embodiments, the anti-thrombotic agent comprises ananticoagulant agent. In certain embodiments, the anticoagulant agentcomprises heparin, a dextran, In certain embodiments the anti-thromboticagent comprises a platelet-inhibitor such as ascorbic acid.

In certain embodiments, the anticoagulant agent comprises heparin.Effective concentrations of heparin for preventing coagulation are knownin the art. In some embodiments, the heparin is present in the undilutedwhole blood sample or the suspension comprising the whole blood sampleat a concentration from about 0.5 IU/mL to about 150 IU/mL at thebeginning of contacting the surface of the acellular tubular scaffold.In certain embodiments, the concentration of heparin at the beginning ofthe contacting is about 5 to about 50 IU/ml. In certain embodiments, theconcentration of heparin at the beginning of the contacting is about 5,about 6, about 7, about 8, about 9, about 10, about 15, or about 20IU/mL. In certain embodiments, the concentration of heparin at thebeginning of the contacting is about 6.7 IU/ml. In some embodiments thebioreactor with tubing and vein is preflushed with a 50 IU/ml heparin inPBS solution (for example for 1 hour), then emptied and filled withblood solution; and in various embodiments, a 1:10 dilution is obtainedby the switching of solutions resulting in around 5 IU/ml heparin in theblood solution. In certain embodiments vacutainers containing 17 IUheparin/mL whole blood are used resulting in about 8.5 IU heparin in theblood solution.

In certain embodiments, the anticoagulant agent comprises a dextran.Dextran of any average molecular weight can be used. In certainembodiments, the dextran has an average molecular weight of about 40 kD,i.e., the dextran is dextran-40. In certain embodiments, the dextran hasan average molecular weight of about 60 kD, i.e., the dextran isdextran-60. In certain embodiments, the dextran has an average molecularweight of about 70 kD, i.e., the dextran is dextran-70. In certainembodiments, the concentration of the dextran at the beginning of thecontacting is about 1 to about 55 g/L. In certain embodiments, theconcentration of the dextran at the beginning of the contacting is about1, about 2, about 3, about 4, about 4.5, about 5, about 10, or about 20g/L. In certain embodiments, the concentration of the dextran at thebeginning of the contacting is about 5 g/L.

In certain embodiments, the anti-thrombotic agent comprises ascorbicacid. In certain embodiments, the concentration of ascorbic acid at thebeginning of the contacting is about 0.2 to about 200 μg/ml. In certainembodiments, the concentration of ascorbic acid at the beginning of thecontacting is about 1, about 5, about 10, about 20, about 50, or about100 μg/mL. In certain embodiments, the concentration of ascorbic acid atthe beginning of the contacting is about 5 μg/mL.

Two or more anti-thrombotic agents (e.g., anticoagulant agents) can becombined in the present invention. In certain embodiments, thesuspension comprises two or more of the agents selected from the groupconsisting of heparin, dextran (e.g., dextran-40), and ascorbic acid. Incertain embodiments, the suspension comprises heparin, dextran (e.g.,dextran-40), and ascorbic acid. The skilled person would appreciate thatwhen two or more anti-thrombotic agents are combined, at least one ofthem may be used at a lower concentration than the concentration atwhich it would be used alone. In certain embodiments, the suspensioncomprises about 6.7 IU/mL of heparin, about 6.7 mg/mL of dextran-40, andabout 4.5 μg/mL of ascorbic acid. In certain embodiments, the suspensioncomprised at least about 6.7 IU/mL of heparin, about 6.7 mg/mL ofdextran-40, and about 4.5 μg/mL of ascorbic acid.

In certain embodiments, the suspension with whole blood or diluted wholeblood or the undiluted whole blood further comprises a growth factorselected from the group consisting of: granulocyte macrophage-colonystimulating factor (GM-CSF), interleukin (IL)-3, IL-4, neutrophin(NT)-6, pleiotrophin (HB-GAM), midkine (MK), interferon inducibleprotein-10 (IP-10), platelet factor (PF)-4, monocyte chemotacticprotein-1 (MCP-1), RANTES (CCL-5, chemokine (C-C motif) ligand 5), IL-8,IGFs, fibroblast growth factor (FGF)-1, FGF-2, FGF-3, FGF-4, FGF-5,FGF-6, FGF-7, FGF-8, FGF-9, transforming growth factor (TGF)-β, VEGF,platelet-derived growth factor (PDGF)-A, PDGF-B, HB-EGF, hepatocytegrowth factor (HGF), tumor necrosis factor (TNF)-α, insulin-like growthfactor (IGF)-1, and any combination(s) thereof. In certain embodiments,the growth factor is a human growth factor. In certain embodiments, theconcentration of the growth factor is equal to the population averagephysiological level of the growth factor. In certain embodiments, theconcentration of the growth factor is more than the population averagephysiological level of the growth factor. In certain embodiments, thegrowth factor is supplemented as a recombinant protein.

In certain embodiments, the growth factor is a fibroblast growth factor(FGF)-2. In certain embodiments, the FGF-2 is human FGF-2. In certainembodiments, the concentration of the FGF-2 at the beginning of thecontacting is about 0.5 to about 200 ng/ml. In certain embodiments, theconcentration of the FGF-2 at the beginning of the contacting is about10 ng/ml. In certain embodiments, the FGF-2 is supplemented prior to thecontacting at the concentration of about 0.5 to about 200 ng/ml (e.g.,about 10 ng/ml). In certain embodiments, the FGF-2 is recombinant humanFGF-2 (rhFGF-2). In certain embodiments, the concentration of therhFGF-2 at the beginning of the contacting is about 0.5 to about 200ng/ml (e.g., about 10 ng/ml).

In certain embodiments, the growth factor is a vascular endothelialgrowth factor (VEGF). In certain embodiments, the VEGF is human VEGF. Incertain embodiments, the concentration of the VEGF at the beginning ofthe contacting is about 4 to about 800 ng/mL. In certain embodiments,the concentration of the VEGF at the beginning of the contacting isabout 80 ng/ml. In certain embodiments, the VEGF is supplemented priorto the contacting at the concentration of about 4 to about 800 ng/mL(e.g., about 80 ng/mL). In certain embodiments, the VEGF is recombinanthuman VEGF (rhVEGF). In certain embodiments, the concentration of therhVEGF at the beginning of the contacting is about 4 to about 800 ng/mL(e.g., about 80 ng/mL).

In certain embodiments, the suspension with whole blood or diluted wholeblood or the undiluted whole blood further comprises acetylsalicylicacid. In certain embodiments, the concentration of acetylsalicylic acidat the beginning of the contacting is between 0.2 μg/mL and 200 μg/mL;or 0.5 μg/mL and 100 μg/mL; or 0.1 μg/mL and 50 μg/mL; or 1 μg/mL and 25μg/mL; or 1 μg/mL and 10 μg/mL; or 2 μg/mL and 10 μg/mL; or 3 μg/mL and8 μg/mL; or 4 μg/mL and 6 μg/mL. In certain embodiments, theconcentration of acetylsalicylic acid at the beginning of the contactingis about 0.5 μg/mL; or about 1 μg/mL; or about 2 μg/mL; or about 3μg/mL; or about 4 μg/mL; or about 5 μg/mL; or about 6 μg/mL; or about 7μg/mL; or about 8 μg/mL; or about 8 μg/mL; or about 8 μg/mL; or about 9μg/mL; or about 10 μg/mL; or about 25 μg/mL or about 50 μg/mL; or about100 μg/mL; or about 200 μg/mL. In certain embodiments, the concentrationof acetylsalicylic acid at the beginning of the contacting is about 5μg/mL. In certain embodiments, the VEGF is supplemented prior to thecontacting at a concentration between 4 ng/mL and 800 ng/mL; or between10 ng/mL and 400 ng/mL; or between 20 ng/mL and 200 ng/mL; or between 40ng/mL and 200 ng/mL; or between 60 ng/mL and 100 ng/mL; or between 70ng/mL and 90 ng/mL.

In certain embodiments, the concentration of human serum albumin (HSA)at the beginning of the contacting is between 50 mg/mL and 150 mg/mL; orbetween 50 mg/mL and 125 mg/mL; or between 50 mg/mL and 100 mg/mL; orbetween 60 mg/mL and 90 mg/mL; or between 60 mg/mL and 80 mg/mL; orbetween 65 mg/mL and 75 mg/mL. In certain embodiments, the concentrationof human serum albumin at the beginning of the contacting is about 55 toabout 105 mg/m, which is the amount when whole blood is diluted in asolution by 2-fold. In certain embodiments, the concentration of humanserum albumin at the beginning of the contacting is about 45 to about 69mg/mL. In certain embodiments, the concentration of HSA in a solution isabout 70 mg/ml, and the concentration of HSA in human blood is about 45mg/mL, and the whole blood in diluted in the solution, the HSA in thesuspension comprising whole blood is 55-60 mg/mL (i.e., 2-fold dilutionof whole blood in the solution). In certain embodiments, theconcentration of human serum albumin after whole blood is diluted in asolution comprising 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, or 20-fold diluted HSA.

In certain embodiments, the suspension with whole blood or diluted wholeblood or the undiluted whole blood comprises one or more nutrientsand/or growth factors, e.g., at physiological concentrations, to supportsurvival, proliferation, and/or differentiation of cells. In certainembodiments, the nutrients are selected from the group consisting ofmonosaccharides (e.g., D-glucose), amino acids (natural amino acids),vitamins, inorganic ions, trace elements, salts, and any combinationsthereof. In certain embodiments, the amino acid included as a nutrientmay be one or more of: L-ArginineHCl, L-Cystine2HCl, L-CystineHCl H₂O,L-HistidineHCl H₂O, L-Isoleucine, L-Leucine, L-LysineHCl, L-Methionine,L-Phenylalanine, L-Threonine, L-Tryptophan, L-Tyrosine2H₂O, L-Valine,L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine,L-Proline, L-Serine, and/or L-Hydroxyproline. In certain embodiments,the vitamin included as a nutrient may be one or more of:K—Ca-Pantothenate, Choline Chloride, Folic acid, i-Inositol,Niacinamide, Pyridoxal HCl, Pyridoxine HCl, Riboflavin, Thiamine HCl,Biotin, Vitamin B12, Para-aminobenzoic acid, Niacin, Ascorbic acid,a-Tocopherol phosphate, Calciferol, Menadione, Vitamin A. In certainembodiments, the inorganic ion is included in the form of an inorganicsalt including but not limited to CaCl₂, KCl, MgSO₄, NaCl, NaHCO₃,NaHPO₄, KNO₃, NaSeO₃, Ca(NO₃)₂, CuSO₄, NaHPO₄, MgCl₂, Fe(NO₃)₃, CuSO₄,FeSO₄, and/or KH₂PO₄.

In some embodiments, the suspension with whole blood or diluted wholeblood or the undiluted whole blood comprises about 0.7% to about 1.5%salt. In some embodiments, the physiological solution comprises about0.7%, about 0.8%, about 0.9%, or about 1.0% salt.

In certain embodiments, the suspension comprises a CO₂-independentbuffer system. In some embodiments, the CO₂-independent buffer systemcomprises a phosphate buffer system. In some embodiments, theCO₂-independent buffer system comprises a phosphate buffer and sodiumbicarbonate. In certain embodiments, the suspension further comprisesone or more of: D-Glucose, Phenol red, HEPES, Sodium pyruvate,Glutathione (reduced), Hypoxantine.Na, Thymidine, Lipoic acid,Putrescine 2HCl, Bacto-peptone, Thymine, Adenine sulphate,Adenosine-5-triphosphate, Cholesterol, 2-deoxy-D-ribose,Adenosine-5-phosphate, Guanine HCl, Ribose, Sodium acetate, Tween 80,Uracil, and/or Xanthine Na.

In certain embodiments, a non-cellular factor of the whole bloodsuspension promotes cellularization of the acellular tubular scaffold,e.g., by promoting cell survival, proliferation, and/or differentiation.In certain embodiments, the non-cellular factor is a non-cellularcomponent present in the whole blood. In certain embodiments, thenon-cellular blood factor is a factor generated from the whole blood(e.g., from the cells in the whole blood) during the contacting. Incertain embodiments, the non-cellular blood factor promotes hostcompatibility, cellularization or integration of the vessel upongrafting. Non-limiting examples of non-cellular factors of the presentinvention are provided in the following table:

Non-cellular factors Hemoglobin subunit delta Hemoglobin subunit epsilonPlasminogen Hemoglobin subunit alpha Hemoglobin subunit gamma-1 Serumalbumin Alpha-2-macroglobulin Complement C1q subcomponent subunit BHemoglobin subunit beta Thrombospondin-1 Adenylate kinase isoenzyme 1Fibrinogen alpha chain Flavin reductase (NADPH) Complement C3 vonWillebrand factor Fibrinogen beta chain Peroxiredoxin-2Inter-alpha-trypsin inhibitor heavy chain H2 Hemoglobin subunit alphaImmunoglobulin kappa variable 2-30 Apolipoprotein B-100 Fermitin familymember 3 Ceruloplasmin Phosphoinositide phospholipase C Fibrinogen betachain Isoform 2 of Fermitin family homolog 3 Alpha-2-macroglobulinIsoform 2 of Spectrin alpha chain, erythrocytic 1 Protein S100-A8Complement C1qB (Fragment) Fibulin-1 Serum paraoxonase/arylesterase 1Hemopexin Fibrinogen alpha chain Hemoglobin subunit beta Complement C4-BComplement C4-A Isoform Er16 of Ankyrin-1 Catalase Complement C3 GC,vitamin D binding protein Serum albumin Serum albuminFructose-bisphosphate aldolase A Coagulation factor X proteinInter-alpha-trypsin inhibitor heavy chain H2 Vitamin K-dependent proteinS 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMPcyclohydrolase Profilin Peroxiredoxin 2 Complement component 4A vonWillebrand factor Complement component C7 Profilin-1 Isoform 3 ofFibronectin Glyceraldehyde-3-phosphate dehydrogenase CeruloplasminFructose-bisphosphate aldolase Isoform 3 of Latent-transforming growthfactor beta-binding protein 1 Isoform 2 of Tropomyosin alpha-1 chainCatalase Selenium-binding protein 1 Tropomyosin alpha-1 chainThrombospondin-3 Peroxiredoxin-1 Tyrosine-protein kinaseAntithrombin-III GTP-binding nuclear protein Ran Peroxiredoxin-6Chloride intracellular channel protein Complement C5a anaphylatoxinIsoform 4 of Tropomyosin alpha-3 chain Tropomyosin alpha-4 chain Proteinarginine N-methyltransferase 5 Complement component C7 Chlorideintracellular channel protein 1 Carbonyl reductase 3 SPARC Isoform 2 ofUbiquitin carboxyl-terminal hydrolase 14 Vinculin or Isoform 1 ofVinculin C-1-tetrahydrofolate synthase, cytoplasmicMethylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1Tyrosine-protein kinase Heparin cofactor 2 SPARC Isoform 2 ofCoagulation factor IX Ras-related protein Rab-30Serine/threonine-protein kinase OSR1 RAS like proto-oncogene B Isoform 2of Adenylyl cyclase-associated protein 1 PDZ and LIM domain protein 1Coagulation factor IX (Fragment) Talin-1 Fibulin-1 OS = Sus scrofaSerine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A betaisoform (Fragm Isoform 2 of Inter-alpha-trypsin inhibitor heavy chain H3Glyceraldehyde-3-phosphate dehydrogenase (Fragment) Glutathioneperoxidase 3 UMP-CMP kinase Cofilin-1 Peptidyl-prolyl cis-transisomerase A Coagulation factor XIII A chain Serine/threonine-proteinkinase OSR1 RAB7A, member RAS oncogene family 6-phosphofructokinaseSuperoxide dismutase ADP-ribosylation factor 6 Alpha-soluble NSFattachment protein Eukaryotic initiation factor 4A-III Pyruvate kinasePKM Eukaryotic translation initiation factor 5A-1 Gelsolin Ras-relatedprotein Rab-14 Adenylyl cyclase-associated protein Adenylylcyclase-associated protein Pyruvate kinase Gelsolin (Fragment) Pyruvatekinase Peroxiredoxin-6 Isoform 3 of Ras-related protein Rap-1bGlyceraldehyde-3-phosphate dehydrogenase Ras-related protein Rab-6B 26Sproteasome regulatory subunit 6A ATP-dependent 6-phosphofructokinase,liver type AP complex subunit beta Transitional endoplasmic reticulumATPase Ras-related protein Rap-1A Pigment epithelium-derived factorT-complex protein 1 subunit zeta T-complex protein 1 subunit delta

Any one of the agents disclosed above, e.g., anti-thrombotic agents,nutrients, diluent, and CO₂-independent buffer system, can be introducedto the diluent (e.g., the physiological solution), and be mixed into thesuspension upon dilution of the whole blood. Alternatively oradditionally, it can be introduced directly to the whole blood prior todilution, and/or be introduced to the suspension after dilution of thewhole blood.

In certain embodiments, the concentration of a nutrient in thesuspension is monitored and adjusted continuously or regularly. Incertain embodiments, the nutrient is selected from the group consistingof a monosaccharide (e.g., D-glucose), an amino acid (e.g., a naturalamino acid), a vitamin, an inorganic ion, a trace element, and a salt.In a specific embodiment, the concentration of D-glucose in thesuspension is monitored during the contacting, and D-glucose is added tothe suspension to maintain D-glucose concentration at about 3 to about11 mmol/L.

In certain embodiments, the concentration of D-glucose in the suspensionis monitored by measuring the concentration of D-glucose in a samplecollected from the suspension. In certain embodiments, the concentrationof D-glucose in the suspension is measured regularly, such as twiceevery day, once every day, or once every two days. In certainembodiments, the concentration of D-glucose in the suspension ismonitored by measuring the concentration of D-glucose using a sensorthat is in contact with the suspension. In certain embodiments, thesensor is continuously in contact with the suspension during thecontacting. In certain embodiments, D-glucose is added when the measuredconcentration of D-glucose in the suspension is below 4 mmol/L. Incertain embodiments, D-glucose is added to reach a final concentrationof about 7 mmol/L; about 8 mmol/L; about 9 mmol/l; or about 10 mmol/L ofD-glucose in the suspension. In some embodiments, the glucose levels areadjusted by addition of a 1.1 M D-glucose stock solution. In certainembodiments addition of a 0.91 μL stock solution (at 1.1M) per mL bloodsolution leads to an increase of 1 mmol/L in the glucose level.Continuous monitoring by a sensor, optionally in combination withautomatic addition of D-glucose in the suspension, allows maintenance ofD-glucose concentration in a narrower ranger. Accordingly, in certainembodiments, D-glucose concentration is maintained at about 5 to about 8mmol/L.

Non-Cellular Blood Factors

In some embodiments, the whole blood sample comprises one or morenon-cellular factors, wherein one or more non-cellular factors of thewhole blood populate the scaffold, and wherein the one or morenon-cellular factors promote cellularization of the acellular tubularscaffold and host compatibility of the vessel upon grafting. As usedherein, the term “non-cellular factor” refers to a component in thewhole blood suspension that does not comprise a complete eukaryoticcell. A non-cellular factor can be any type of molecule, including butnot limited to protein, nucleic acid, saccharide, lipid, small molecule,metal ion, or a conjugate or combination thereof.

For example, the non-cellular factor may be growth factors. In someembodiments, the growth factor is selected from the group consisting of:granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin(IL)-3, IL-4, neutrophin (NT)-6, pleiotrophin (HB-GAM), midkine (MK),interferon inducible protein-10 (IP-10), platelet factor (PF)-4,monocyte chemotactic protein-1 (MCP-1), RANTES (CCL-5, chemokine (C-Cmotif) ligand 5), IL-8, IGFs, fibroblast growth factor (FGF)-1, FGF-2,FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, transforming growthfactor (TGF)-β, VEGF, platelet-derived growth factor (PDGF)-A, PDGF-B,HB-EGF, hepatocyte growth factor (HGF), tumor necrosis factor (TNF)-α,insulin-like growth factor (IGF)-1, and any combination(s) thereof.

In certain embodiments, the non-cellular blood factor is a cell fragment(e.g. thrombocyte, cell fraction). In certain embodiments, thenon-cellular blood factor is a protein (e.g., cytokine, chemokine,growth factor, antibody, antibody fragment, protein of the complementsystem, or enzyme). In certain embodiments, the non-cellular bloodfactor is a nucleic acid (e.g., circulating DNA, circulating RNA, orcirculating miRNA). In certain embodiments, the non-cellular bloodfactor is a saccharide (e.g., monosaccharide or disaccharide). Incertain embodiments, the non-cellular blood factor is a lipid (e.g.,fatty acid, triglyceride, or cholesterol). In certain embodiments, thenon-cellular blood factor is a small molecule. In certain embodiments,the non-cellular blood factor is metal ion.

In certain embodiments, the non-cellular blood factor is a modified formor a metabolite of any one of the molecules above. For example, incertain embodiments, the non-cellular blood factor is a segment of aprotein (e.g., a peptide or polypeptide). In certain embodiments, thenon-cellular blood factor is a nucleic acid with one or moremodifications (e.g., methylation, 5′ capping). In certain embodiments,the non-cellular blood factor is an aldonic acid or alditol of asaccharide. In certain embodiments, the non-cellular blood factor is asterol. In certain embodiments, the non-cellular blood factor is aconjugate of any one of the molecules disclosed herein, wherein themolecules are conjugated covalently or noncovalently.

In certain embodiments, the concentration of the growth factor is equalto the population average physiological level of the growth factor. Incertain embodiments, the concentration of the growth factor is more thanthe population average physiological level of the growth factor. Incertain embodiments, the growth factor is supplemented as an isolated,purified, and/or synthetic molecule.

The non-cellular blood factor can contribute to thereconditioning/recellularization of the acellular tubular scaffold invarious ways in vitro or after implantation. For example, in certainembodiments, the non-cellular blood factor promotes attachment oradherence of cells to the scaffold. In certain embodiments, thenon-cellular blood factor promotes proliferation of progenitor cells. Incertain embodiments, the non-cellular blood factor promotesdifferentiation of progenitor cells into cells that can populate thescaffold. In certain embodiments, the non-cellular blood factor inhibitsapoptosis and/or necrosis of the cells and/or progenitor cells. Incertain embodiments, the non-cellular blood factor promotes removal ormasking of allogeneic protein(s) present on the scaffold. In certainembodiments, the non-cellular blood factor promotes deposit ofautologous protein(s) on the scaffold. In certain embodiments, thenon-cellular blood factor promotes conditioning of the scaffold tosuppress host rejection.

Contacting the Acellular Tubular Scaffold with Patient Whole Blood

The present disclosure provides a method of preparing a personalizedblood vessel, the method comprising contacting the surface (e.g., innersurface) of an acellular tubular scaffold (e.g., a decellularized bloodvessel or a bioprinted tubular scaffold) with a suspension comprisingwhole blood for more than 48 hours, thereby allowing the cells presentin the blood to populate the scaffold. In some embodiments, the surfaceof the acellular tubular scaffold is the inner surface of the acellulartubular scaffold.

As used herein, the term “contact,” contacting,” or contacted” meanstatic incubation, perfusion, introducing, injecting, or culturing.Perfusion may be pulsed perfusion, interval perfusion, alternatingdirection, and no perfusion. In some embodiments, the acellular tubularscaffold is continuously perfused with the suspension or whole blood.

In certain embodiments, the contacting is for more than 2 days. As usedherein, the term “more than 2 days” refers to a duration that is longerthan 48 hours and longer than about 48 hours. The terms “day” and “days”refer to 24 hour cycle(s). In certain embodiments, the contacting is forat least 3, at least 4, at least 5, at least 6, or at least 7 days. Incertain embodiments, the contacting is for up to 9, up to 10, up to 11,up to 12, up to 13, up to 14 days, up to 21 days, or up to 31 days. Incertain embodiments, the contacting is for up to 1 month. In someembodiments, the contacting is for from about 1 to 31 days, for 2 to 21days, for 2 to 14 days, for 2 to 9 days, for 2 to 7 days, for 2 to 5days, for 5 to 7 days, or for 7 to 9 days. In certain embodiments, thecontacting is for about 7 to about 9 days. In some embodiments,contacting the surface of the acellular tubular scaffold is for 2 to 21days, for 3 to 21 days, for 4 to 21 days, for 5 to 21 days, for 6 to 21days, or for 7 to 21 days. In certain embodiments, the contacting is forabout 2 days, for about 3 days, for about 4 days, for about 5 days, forabout 6 days for about 7 days, for about 8 days, for about 9 days, forabout 10 days, for about 11 days, for about 12 days, for about 13 days,for about 14 days, for about 15 days, for about 16 days, for about 17days, for about 18 days, for about 19 days, for about 20 days, or forabout 21 days.

The acellular tubular scaffold (e.g., a decellularized blood vessel or abioprinted tubular scaffold) may be contacted with undiluted whole bloodsample or the suspension containing diluted whole blood in any mannerknown in the art. For example, in certain embodiments, the scaffold isloose and suspended in the suspension, optionally wherein the suspensionis shaken (e.g., rotated) to re-mix the components regularly orcontinuously. Rotation of the tubular scaffold during perfusion may becontinuous/intermittent, alternating, or interval.

In certain embodiments, the inner surface of the scaffold is contactedwith the suspension. In certain embodiments, the scaffold is perfusedwith the suspension. In certain embodiments, the scaffold is perfusedwith a closed recirculation of the suspension. In certain embodiments,the continuous contacting is perfusion conducted using a bioreactor. Incertain embodiments, the outer surface of the scaffold is also contactedwith the suspension. In other embodiments, the outer surface of thescaffold is not contacted with the suspension. In certain embodiments,the contacting is conducted in vitro.

The perfusion can be conducted in any mode. For example, in certainembodiments, the scaffold is perfused with the suspension continuously.In certain embodiments, the scaffold is perfused with the suspensionintermittently (e.g., perfusion for 1 minute at an interval of 4minutes). In certain embodiments, the perfusion is conducted at a singledirection. In certain embodiments, the single direction is the directionof antegrade flow, e.g., the direction allowed by the valve structure(s)in the scaffold. In certain embodiments, the perfusion is conducted atalternating directions (e.g., changing direction every 5 minutes). Thespeed of the perfusion can be determined by the skilled person in lightof a number of factors, such as the diameter of the tubular scaffold,the mechanical strength of the scaffold, the time taken for the cells tosediment, and the viscosity of the suspension. In certain embodiments,the scaffold is perfused at a speed of about 0.1 to about 50 mL perminute. In certain embodiments, the scaffold is perfused at a speed ofabout 2 ml per minute. Depending on the orientation of the scaffold,rotation during the perfusion (including the intervals) may beadvantageous such that the inner surface of the scaffold is populatedevenly. In certain embodiments, the scaffold is rotated continuously. Incertain embodiments, the scaffold is rotated intermittently (e.g., by30° every 5 minutes). The direction of the rotation may be constant oralternating. The advantage of rotation may not be substantially wherethe scaffold is fixed vertically. Accordingly, in certain embodiments,the scaffold is not rotated.

In specific embodiments, the scaffold (e.g., the lumen of the scaffold)is continuously perfused with the suspension. In certain embodiments,the scaffold is perfused at a speed of about 0.1 to about 50 mL perminute (e.g., about 2 mL per minute). In certain embodiments, thescaffold is perfused with a closed recirculation of the suspension. Incertain embodiments, the continuous contacting is perfusion conductedusing a bioreactor.

Monitoring Environmental Conditions During the Contacting

In another aspect, the present disclosure provides a method of preparinga personalized blood vessel, the method comprising contacting thesurface of an acellular tubular scaffold (e.g., a decellularized bloodvessel or a bioprinted tubular scaffold) with a suspension comprisingwhole blood, wherein a plurality environmental parameters (e.g.,temperature, pH, oxygen content, and/or CO₂ content) are monitored andadjusted continuously or regularly.

In certain embodiments, the contacting is conducted at about 8° C. toabout 40° C. In certain embodiments, the contacting is conducted atabout 20° C. to about 25° C. In certain embodiments, the contacting isconducted at about 37° C. In certain embodiments, the contacting isconducted at about 20° C., about 21° C., about 22° C., about 23° C.,about 24° C., or about 25° C.

In certain embodiments, the contacting is conducted at about pH 7.2 toabout pH 7.5. In certain embodiments, the contacting is conducted atabout pH 7.4.

In certain embodiments, the contacting is conducted at about 30 mmHg toabout 100 mmHg of partial pressure of oxygen. In certain embodiments,the contacting is conducted at about 30 mmHg to about 40 mmHg of partialpressure of oxygen. In certain embodiments, the contacting is conductedat about 80 mmHg to 100 mmHg of partial pressure of oxygen. In certainembodiments, the contacting is conducted at about 160 mmHg of partialpressure of oxygen.

In certain embodiments, the contacting is conducted at about 38 mmHg toabout 76 mmHg of partial pressure of carbon dioxide. In certainembodiments, the contacting is conducted at about 38 mmHg of partialpressure of carbon dioxide. In certain embodiments, the contacting isconducted at about 76 mmHg of partial pressure of carbon dioxide. Incertain embodiments, the contacting is conducted at about 40 mmHg toabout 50 mmHg in of partial pressure of carbon dioxide.

In certain embodiments, the environmental parameters (e.g., temperature,pH, oxygen content, and/or CO₂ content) are monitored and adjustedcontinuously or regularly during the contacting.

In another aspect, the present disclosure provides a method of preparinga personalized blood vessel, the method comprising contacting thesurface of an acellular tubular scaffold (e.g., a decellularized bloodvessel or a bioprinted tubular scaffold) with a suspension comprisingwhole blood, wherein the concentration of a nutrient in the suspensionis monitored and adjusted continuously or regularly.

In another aspect, the present disclosure provides a method of preparinga personalized blood vessel, the method comprising contacting thesurface of an acellular tubular scaffold (e.g., a decellularized bloodvessel or a bioprinted tubular scaffold) with a suspension comprisingwhole blood, wherein the concentration of D-glucose in the suspension ismonitored during the contacting, and D-glucose is added to thesuspension to maintain D-glucose concentration at about 3 to about 11mmol/L.

In some embodiments, the concentration of D-glucose in the suspension ismonitored by measuring the concentration of D-glucose in a samplecollected from the suspension. In some embodiments, the concentration ofD-glucose in the suspension is measured once every day. In someembodiments, the concentration of D-glucose in the suspension ismonitored by measuring the concentration of D-glucose using a sensorthat is in contact with the suspension. In some embodiments, the sensoris continuously in contact with the suspension during the contacting.

In some embodiments, D-glucose is added when the measured concentrationof D-glucose in the suspension is below 4 mmol/L. In some embodiments,D-glucose is added to reach a final concentration of 10 mmol/L ofD-glucose in the suspension.

Preparation of Acellular Tubular Scaffold with an Enriched or SelectedComponent of Whole Blood

In certain embodiments, the present disclosure provides a method ofpreparing a personalized blood vessel in which one or more components ofwhole blood is enriched and selected, and then added to the surface ofan acellular tubular scaffold to contact with the inner surface of thescaffold. For example, a component selected from thrombocytes, nucleatedcells, proteins, growth factors, signaling factors, immunoglobulins, andany combination(s) thereof, can be added to the inner surface of anacellular tubular scaffold. The component may be enriched or selectedby, for example, centrifugation, gradient centrifugation, separation byselective adhesion, filtration, or sorting (e.g., FACS, MACS).

The terms “enriched and selected” refers to a substance and/or entitythat has been separated from at least one of the components with whichit was mixed when initially produced (whether in nature or in anexperimental setting). In certain embodiments, an isolated substanceand/or entity is separated from at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99% free of the componentswith which it was mixed when initially produced. In certain embodiments,an enriched and selected substance and/or entity are purified. Incertain embodiments, an enriched, selected, and/or purified substanceand/or entity is substantially free of other components. As used herein,calculation of percent separation or purity does not include excipients(e.g., buffer, solvent, water, etc.).

Personalized Blood Vessels

The method disclosed herein allows cells and/or progenitor cells topopulation the surface (e.g., inner surface) of the acellular tubularscaffold (e.g., a decellularized blood vessel or a bioprinted tubularscaffold), thereby generating the personalized blood vessel. In certainembodiments, the cells comprise endothelial cells. In certainembodiments, the cells comprise smooth muscle cells. In certainembodiments, the progenitor cells comprise endothelial progenitor cells.In certain embodiments, the progenitor cells comprise smooth muscleprogenitor cells. The skilled person would understand that the cellsand/or progenitor cells might populate the scaffold in one or more ways.Population of the scaffold can occur in vitro prior to implantation orin vivo after implantation. In certain embodiments, the cells attach tothe scaffold. In certain embodiments, the progenitor cells proliferateand/or differentiate to generate progeny cells, and the progeny cellsattach to the scaffold.

In certain embodiments, the contacting results in proliferation and/ordifferentiation of the progenitor cells to endothelial cells. In certainembodiments, the endothelial cells express VE-cadherin, AcLDL, vWF,and/or CD31. The reconditioned/recellularized scaffold can becharacterized for presence of endothelial and smooth muscle cells.Immunohistochemistry and immunofluorescence techniques well known to theordinarily skilled artisan are utilized to detect the presence orabsence of endothelial and smooth muscle cells. To visualize thepresence of endothelial cells, antibodies to CD31 (1:200) (Abcam,Germany) and vWF (1:100) (Santa Cruz, Germany) can be used for stainingof the reconditioned/recellularized scaffolds. To visualize smoothmuscle cells, antibody against smooth muscle actin (1:50) (Abcam,Germany) can be used to stain the reconditioned/recellularized valves.Presence of cells positive of these markers in the recellularizedscaffolds are detected by immunohistochemistry or immunofluorescence.Smooth muscle cells can also be identified by the morphology ofspindle-shaped muscle cells lining the reconditioned/recellularizedscaffolds.

The personalized blood vessels prepared by the method disclosed hereinare useful for implantation into a subject (e.g., human). In certainembodiments, the personalized blood vessel is useful as a vein. Incertain embodiments, the method further comprises a step of assessingthe venous valve function (e.g., using an in vitro valve competencetest) of the personalized blood vessel. Exemplary in vitro valvecompetence tests are provided in U.S. Patent Publication No.2017/0071738. In certain embodiments, the venous valve function istested before reconditioning/recellularization. In certain embodiments,the venous valve function is tested afterreconditioning/recellularization. In certain embodiments, the venousvalve function is tested both before and afterreconditioning/recellularization.

A functional personalized blood vessel has no leakage. Accordingly, incertain embodiments, the method further comprises a step of assessingleakage. Various methods are known in the art for testing leakage. Forexample, in certain embodiments, the personalized blood vessel isflushed with a solution (e.g., a physiologically buffered solution suchas phosphate buffered saline (PBS)), preferably at a steady rate, andthe surface of the personalized blood vessel is observed foraccumulation of solution resulting from a leak or hole. In certainembodiments, leakage is tested before reconditioning/recellularization.In certain embodiments, leakage is tested afterreconditioning/recellularization. In certain embodiments, leakage istested both before and after reconditioning/recellularization.

In another aspect, the present disclosure provides a personalized bloodvessel prepared by any one of the methods disclosed herein. In certainembodiments, the personalized blood vessel comprises an acellulartubular scaffold (e.g., a decellularized blood vessel, optionallyMMP-treated, or a bioprinted tubular scaffold) including non-cellularfactors such as ECM components/composition of a mammalian blood vesselor a functional part thereof, cellularized with human cells (e.g.,endothelial cells and/or smooth muscle cells).

The personalized blood vessel of the present disclosure furthercomprises cells such as, but not limited to, whole blood derived stem orprogenitor cells such as endothelial stem cells, endothelial progenitorcells, smooth muscle progenitor cells, whole blood, peripheral blood,and any cell populations that can be isolated from whole blood. Theprogenitor cells are defined as cells that are committed todifferentiate into one type of cells. For example, endothelialprogenitor cells mean cells that are programmed to differentiate intoendothelial cells; smooth muscle progenitor cells means cells that areprogrammed to differentiate into smooth muscle cells. Progenitor cellsin whole blood or peripheral blood includes population of uncommittedand/or committed cells, such as pluripotent cells or totipotent cells.

In certain embodiments, the surface of the personalized blood vessel issubstantially covered by cells. In certain embodiments, the innersurface of the personalized blood vessel is substantially covered byendothelial cells. In certain embodiments, the endothelial cells expressVE-cadherin, AcLDL, vWF, and/or CD31.

The skilled person would appreciate that the cells and/or progenitorcells in the blood need not populate the inner surface of the scaffoldto the extent that the reconditioned/recellularized scaffold isindistinguishable from native blood vessels. As disclosed herein,recellularization can occur in vitro as well as in vivopost-implantation, and a scaffold partially recellularized in vitro canbe further recellularized in vivo to recapture the morphology and/orfunction of native blood vessels. Notwithstanding, endothelial cells ata lower density than in native blood vessels may also substantiallycover the inner surface of the scaffold by adopting a more spreadmorphology, thereby attaching to a larger surface area per cell.

The personalized blood vessels prepared by the method disclosed hereinare useful for implantation into a subject (e.g., human). In certainembodiments, the personalized blood vessel is useful as a vein.

In certain embodiments, the acellular tubular scaffold is derived from avein (e.g., prepared by decellularizing a native vein). In certainembodiments, the vein is in a lower extremity. The venous system of thelower extremities includes the deep veins, which lie beneath themuscular fascia and drain the lower extremity muscles; the superficialveins, which are above the deep fascia and drain the cutaneousmicrocirculation; and the perforating veins that penetrate the muscularfascia and connect the superficial and deep veins. Communicating veinsconnect veins within the same compartment.

In certain embodiments, the vein is a deep vein. Deep veins useful forthe instant methods include but are not limited to superficial femoralvein, which connects the popliteal vein to the common femoral vein, anddeep veins of the calf (e.g., anterior tibial, posterior tibial, andperoneal veins). In certain embodiments, the vein is a supervicial vein.Deep veins useful for the instant methods include but are not limited togreat saphenous vein, the anterior and posterior accessory greatsaphenous veins, small saphenous vein (SSV), and intersaphenous vein(a.k.a. Giacomini vein).

Most veins have venous valves to assure unidirectional flow of blood.For example, the superficial, deep, and most perforating veins in alower extremity contain bicuspid valves. Accordingly, in certainembodiments, the acellular tubular scaffold comprises a valve structure,and the valve structure is reconditioned/recellularized to provide afunctional venous valve in the personalized blood vessel. The valvestructure can be derived either from a native venous valve or from thescaffold synthesis and/or assembly. Methods of assessing the function ofa venous valve are known in the art, including but not limited to an invitro valve competence test. In certain embodiments, the method furthercomprises assessing the venous valve function (e.g., using an in vitrovalve competence test) of the acellular tubular scaffold.

In certain embodiments, the valve structure in the acellular tubularscaffold is reconditioned/recellularized during thereconditioning/recellularization of the rest of the inner surface of thescaffold, i.e., no additional step is taken to specificallyrecondition/recellularize the valve structure. In certain embodiments,the reconditioned/recellularized valves are CD31-positive. Thereconditioned/recellularized valves can also be smooth muscleactin-positive, vWF-positive, and/or be characterized by the presence ofspindle-shaped smooth muscle cells.

In certain embodiments, the personalized blood vessel had one or morefeatures of a native blood vessel. In certain embodiments, thepersonalized vein prepared by a method disclosed herein comprisesreconditioned/recellularized valves have mechanical properties of forceat first peak above 0.8 N. In certain embodiments, the personalizedblood vessel prepared by a method disclosed herein demonstrates noleakage when tested.

Methods of Treatment or Use

In another aspect, the present disclosure provides a personalized bloodvessel disclosed herein for use in therapy. In certain embodiments, thepresent disclosure provides the personalized blood vessel for use intransplantation in a subject. In certain embodiments, the presentdisclosure provides the personalized blood vessel for use in treating ablood vessel disease or disorder of a subject in need thereof.

The terms “treat,” “treating,” or “treatment,” and other grammaticalequivalents as used in this disclosure, include alleviating, abating,ameliorating, or preventing a disease, condition or symptoms, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, inhibiting the disease or condition, e.g., arrestingthe development of the disease or condition, relieving the disease orcondition, causing regression of the disease or condition, relieving acondition caused by the disease or condition, or stopping the symptomsof the disease or condition, and are intended to include prophylaxis.The terms further include achieving a therapeutic benefit and/or aprophylactic benefit. By therapeutic benefit is meant eradication oramelioration of the underlying disorder being treated. Also, atherapeutic benefit is achieved with the eradication or amelioration ofone or more of the physiological symptoms associated with the underlyingdisorder such that an improvement is observed in the patient,notwithstanding that the patient may still be afflicted with theunderlying disorder.

The methods disclosed herein are particularly suitable for generatingautologous-like engineered blood vessels. They have the advantages of:(1) being non-immunogenic and therefore having minimal risk of graftrejection or adverse immune response; (2) obviating the need forimmunosuppression, and therefore less risk to the patient after surgeryand for their lifetime; (3) having no length restriction; (4) being morereadily available, as compared to matched donor veins or autologousveins; (5) being composed of natural components (i.e., ECM, endothelialcells and smooth muscle cells), and therefore having superior qualitiesto mostly synthetic and artificial veins, including preserving residualangiogenic growth factors and biomechanical integrity; 6) requiringinvasive production of vein in comparison to harvesting autologous veinfor transplant; (7) allowing rapid and minimally invasive procedure tosubject by use of whole blood; (8) becoming cellularized andbiologically integrated into the subject's body and it's functions(repair, growth, immune-defense).

Accordingly, in one aspect, the present disclosure provides a method ofsurgery comprising implanting the personalized blood vessel disclosedherein into a subject (e.g., human) in need thereof. In another aspect,the present disclosure provides the personalized blood vessel disclosedherein for use in implantation into a subject (e.g., human) in needthereof. In another aspect, the present disclosure provides apersonalized blood vessel prepared by any one of the methods disclosedherein, wherein the personalized blood vessel has been implanted into asubject (e.g., human) by surgery.

In certain embodiments, the personalized blood vessel is autologous. Asused herein, the term “autologous” means the blood used in the method ofpreparing a personalized blood vessel is from the subject receiving theimplantation or surgery. In certain embodiments, wherein thepersonalized blood vessel is produced by decellularizing a native bloodvessel, the donor of the native blood vessel is not the same individualas the recipient of the personalized blood vessel. In certainembodiments, wherein the personalized blood vessel is produced bydecellularizing a native blood vessel, the native blood vessel isobtained from a suitable animal species (e.g., pig, sheep, cow).

The methods of surgery disclosed herein are useful for treating variousvascular diseases or disorders. Accordingly, in certain embodiments, thesubject has a vascular disease or disorder, e.g., a venous disease ordisorder. In certain embodiments, the venous disease or disorder isselected from the group consisting of deep vein thrombosis (DVT),chronic venous insufficiency (CVI) (a.k.a. postphlebitic syndrome),varicose veins, venous ulceration (e.g., venous leg ulceration), andrecurrent leg cancer (e.g., caused by deep venous reflux and/or venoushypertension). In certain embodiments, the personalized blood vessel isimplanted, transplanted, or grafted to replace a segment of native bloodvessel afflicted with any one of the vascular diseases or disorders. Incertain embodiments, the personalized blood vessel is a vein andcomprises at least one venous valve.

In certain embodiments, the method of surgery treats, ameliorates,and/or palliates one or more symptoms including dull aching, heaviness,or cramping in legs, itching and tingling, pain that gets worse whenstanding, pain that gets better when legs are raised, swelling of thelegs, redness of the legs and ankles, skin color changes around theankles, varicose veins on the surface (superficial), thickening andhardening of the skin on the legs and ankles (lipodermatosclerosis),ulcers on the legs and ankles, and wound that is slow to heal on thelegs or ankles.

In certain embodiments, the method of surgery disclosed herein treatsand/or ameliorates CVI. CVI defines those manifestations of venousdisease resulting from ambulatory venous hypertension, defined as afailure to reduce venous pressure with exercise. Under normalcircumstances, the venous valves and the muscular pumps of the lowerextremity limit the accumulation of blood in the lower extremity veins.Failure of the lower extremity muscle pumps due to out-flow obstruction,musculo-fascial weakness, loss of joint motion, or valvular failure isassociated with peripheral venous insufficiency.

In certain embodiments, the method of surgery disclosed herein treatsand/or ameliorates venous ulceration. Venous ulcerations are wounds dueto improper functioning of the venous valves, usually of the legs (i.e.,venous leg ulceration). Venous ulcers arise when valves have reducedfunction and the backflow of blood causes pooling of blood in the veinsand increased pressure in the veins and capillaries. This leads to otherrelated complications, such as edema, inflammation, hardening of thetissue, malnutrition of the skin, and venous eczema. Venous ulcers arelarge, shallow, discolored due to leakage of iron-containing pigment inred blood cells into the tissue, and may have discharge. The ulcers aremost frequently situated around the medial or later malleoli.

In certain embodiments, the method of surgery disclosed herein restoresnormal lower extremity venous pressure. For example, with walking, lowerextremity venous pressure is reduced from approximately 100 mm Hg(depending on height) to mean of 18 mm Hg to about 25 mm Hg within 7 to12 steps. Similar pressure changes are observed with standing ankleplanter flexion or heel rising, transferring weight to the forefoot (thetiptoe maneuver). Ambulatory venous pressure (AVP) can be determinedusing a 21-gauge needle to measure the response to 10 tiptoe movements,usually at a rate of 1 per second, in dorsal foot vein. When resuming astating standing position, hydrostatic pressure is restored after a meanof 31 seconds. The incidence of ulceration has a linear relationship toincreases in AVP above 30 mm Hg. An increased AVP is also associatedwith a 90% venous refill time of less than 20 seconds. In contrast tothe AVP, volume changes can be measure non-invasively usingplethysmography. Rapid reflux (i.e., venous filling of greater than 7ml/sec) and calf pump dysfunction are associated with a high incidenceof ulceration. The reconditioned/recellularized valves in veins, upongrafting, restore the normal AVP, normal rapid reflux and/or normal calfpump dysfunction. Preferably, a venous valve in the personalized bloodvessel, upon grafting, restores the normal tolerance of reflux pressureof about 100 mm Hg, and reduces to a mean of about 18 mm Hg to about 25mm Hg during walking 7 to 12 steps.

In certain embodiments, the method of surgery disclosed herein treatsand/or ameliorates the symptoms of incompetent valves in the thigh. Incertain embodiments, the treatment and/or amelioration of the symptomsis achieved by restoring normal working relationship between musclepumps and the venous valves. The muscular pumps of the lower limbinclude those of the foot, calf, and thigh. Among these, the calf pumpis the most important as it is most efficient, has the largestcapacitance and generates the highest pressures (200 mm of mercuryduring muscular contraction). The normal limb has a calf volume rangingfrom 1500 to 3000 cc, a venous volume of 100 to 150 cc, and ejects over40% to 60% of the venous volume with a single contraction.

During contraction, the gastrocnemius and soleus muscles drive bloodinto the large capacity popliteal and femoral veins. Thereconditioned/recellularized valves of the present disclosure preventretrograde flow (reflux) during subsequent relaxation, generatingnegative pressure and drawing blood from the superficial to the deepsystem through competent perforating veins. Thereconditioned/recellularized valves incrementally lower venous pressureuntil arterial inflow equals venous outflow. The present disclosureprovides that when exercise ceases in a subject, the veins withreconditioned/recellularized valves slowly fill the capillary bed,causing a slow return to the resting venous pressure.

Although muscle surrounds the thigh veins, the contribution of thighmuscle contraction to venous return is minimal compared with the calfmuscle pump. Pumping action due to compression of the planter venousplexus during ambulation primes the calf pump. Various leg pumps worktogether with competent valve function to return venous blood from thedistal to proximal extremity. The personalized blood vessels of thepresent disclosure are for use in restore functional leg pumps to returnvenous blood from the distal to proximal extremity.

Bioreactors

In another aspect, the present disclosure provides a bioreactor forpreparing a personalized blood vessel, the bioreactor comprising a pump(for example a peristaltic pump, a gravity pump, a plunger pump, orother suitable pump), a container, a first connector, and a secondconnector, wherein the first and second connectors are directly orindirectly connected to the container, wherein each connector isconnected to an end of a tubular scaffold for preparing a personalizedblood vessel prepared by any one of the methods of preparing apersonalized blood vessel disclosed herein, wherein when the first andsecond connectors are connected to the two ends of a tubular scaffold,the pump mediates the circulation of a suspension, whole blood, orsolution in a closed circuit. In many embodiments it is advantageous ofa pump (for example a peristaltic pump, a gravity pump, a plunger pump,or other suitable pump) used herein is sufficiently gentle so as tominimize damage to blood cells.

In certain embodiments, the bioreactor further comprises a tubularscaffold (e.g., an acellular tubular scaffold (e.g., a decellularizedblood vessel or a bioprinted tubular scaffold)). In certain embodiments,the bioreactor further comprises a suspension or whole blood asdisclosed herein for preparing a personalized blood vessel.

In certain embodiments, at least two parts of the bioreactor areprovided separately, with an instruction to connect them in theprescribed order. Accordingly, in one aspect, the present disclosureprovides a kit for assembling a bioreactor for preparing a personalizedblood vessel, the bioreactor comprising a peristaltic pump, a container,a first connector, and a second connector, wherein the first and secondconnectors can be directly or indirectly connected to the container,wherein each connector can be connected to an end of a tubular scaffoldfor preparing a personalized blood vessel prepared by any one of themethods of preparing a personalized blood vessel disclosed herein,wherein when the first and second connectors are connected to the twoends of a tubular scaffold, the peristaltic pump mediates thecirculation of a suspension, whole blood, or solution in a closedcircuit. In certain embodiments, the kit further comprises a tubularscaffold (e.g., an acellular tubular scaffold (e.g., a decellularizedblood vessel or a bioprinted tubular scaffold)).

In certain embodiments, the first and second connectors are Luerconnectors. In certain embodiments, the first container is directlyconnected to the container by a tube, and/or the second container isdirectly connected to the container by a tube.

In certain embodiments, the bioreactor comprises a sampling port. Incertain embodiments, the sampling port is a part of the container. Incertain embodiments, the sampling port allows withdrawal of a sample ofthe suspension, whole blood, or solution from the closed circuit. Incertain embodiments, the sampling port comprises a sensor for measuringthe temperature, pH, and/or the concentration of oxygen, CO₂, ornutrient (e.g., D-glucose) in the suspension, whole blood, or solution.

In certain embodiments, the sampling port is also useful as an injectionport. In certain embodiments, the bioreactor further comprises aninjection port. In certain embodiments, the injection port is or can beconnected to a reservoir of oxygen, CO₂, or nutrient (e.g., D-glucose).

In certain embodiments, the outer surface of the tubular scaffold iscontacted with the suspension, whole blood, or solution in thecontainer. This bioreactor allows reconditioning/recellularization ofthe inner surface and the outer surface of the tubular scaffold at thesame time.

In certain embodiments, the bioreactor further comprises a chamberenclosing the tubular scaffold, thereby allowing contacting of the outersurface of the tubular scaffold with a liquid in the chamber. Thechamber can also be sterilized, keeping the tubular scaffold in anaseptic condition.

The skilled person would understand that the same bioreactor could beused for decellularizing a native blood vessel or for conditioning atubular scaffold. The appropriate suspension, whole blood, or solutioncan be selected by the skilled person corresponding to the use.

Various pumps can be used, that do not disrupt the cellular andacellular components in the blood solution even during extended periodsof perfusion. These can be peristaltic pumps, gravity pumps, pistonpumps or similar. The inclusion of sample ports in the bioreactor setupallows sterile sampling of the perfusion medium as well as the sterileinjection of additional components without opening the closed loop andin some cases without even stopping the perfusion. In an improvedversion of the bioreactor setup, sensors a variety of relevantparameters such as glucose, pH, oxygen content, CO2 content, metabolitesand so on can be coupled in-line into the perfusion tubing. This allowscontinuous monitoring of the personalization process during RC or duringDC, monitor the progress and success of cell and DNA disruption andremoval.

Another aspect of the present disclosure relates to a bioreactor forpreparing a personalized blood vessel, the bioreactor comprising aperistaltic pump, a container comprising a sampling port, a firstconnector, and a second connector, wherein the first and secondconnectors are directly or indirectly connected to the container,wherein each connector is connected to an end of a tubular scaffold forpreparing a personalized blood vessel prepared by the methods disclosedherein, wherein when the first and second connectors are connected tothe two ends of a tubular scaffold, the peristaltic pump mediates thecirculation of a suspension or solution in a closed circuit.

In some embodiments, the first and second connectors are Luerconnectors. In some embodiments, the sampling port is an injection port.In some embodiments, the bioreactor further comprises an injection port.In some embodiments, the injection port is connected to a reservoir ofD-glucose. In some embodiments, the first container is directlyconnected to the container by a tube, and/or the second container isdirectly connected to the container by a tube. In some embodiments, thebioreactor comprises one or more sample ports. In some embodiments, thebioreactor further comprises one or more sensors for measuring glucoselevel. In some embodiments, the bioreactor further comprises apH-adjusting module. In some embodiments, the bioreactor furthercomprises a CO₂ adjusting module.

Another aspect of the present disclosure relates to a method ofpreparing a personalized blood vessel, the method comprising contactinga surface of an acellular tubular scaffold with a component contained inthe whole blood, which is enriched or selected prior to use incontacting the surface.

In some embodiments, the component is selected from thrombocytes,nucleated cells, proteins, growth factors, signaling factors,immunoglobulins, and any combinations thereof.

In some embodiments, the component is enriched by centrifugation,gradient centrifugation, separation by selective adhesion, filtration,or sorting. Many methods for enriching or sorting components are wellknown in the art. Some example of methods for sorting includesfluorescence activated cell sorting (FACS), magnetic-activated cellsorting (MACS).

Definitions

It is to be understood that methods are not limited to the particularembodiments described, and as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting. Thescope of the present technology will be limited only by the appendedclaims.

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include singular and plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a singlecell as well as a plurality of cells, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this disclosure.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

The term “about” refers to any minimal alteration in a stated absolutevalue (e.g., the concentration or amount of an agent) that does notchange the stated efficacy, activity, action, results, etc. Inembodiments, the term “about” may include ±10% of a specified numericalvalue or data point. The term “about” includes the stated value (e.g.,“about 1%” includes 1% as well as minimal alterations thereof).

Ranges can be expressed in this disclosure as from “about” a firstparticular value, and/or to “about” a second particular value. When sucha range is expressed, another aspect including from the first particularvalue and/or to the second particular value is also contemplated.Similarly, when values are expressed as approximations, by use of“about,” it is understood that the particular value forms anotheraspect. It is further understood that the endpoints of each of theranges are significant both in relation to the other endpoint, andindependently of the other endpoint. It is also understood that thereare a number of values disclosed in this disclosure, and that each valueis also disclosed as “about” that particular value in addition to thevalue itself. It is also understood that throughout the application,data are provided in a number of different formats and that this datarepresent endpoints and starting points and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this disclosure.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

EXAMPLES Example 1: Personalized Tissue Engineered Vein (P-TEV)

Porcine P-TEV grafts were prepared and tested in vivo to show theirsafety and feasibility. Specifically, pig vena cava was chosen as asubstitute model system to mimic the size and thickness of the vesselwall of a human femoral vein.

Preparation of P-TEV

Vena cava was retrieved from pig cadavers and was decellularized toremove the donor cells and DNA. Briefly, the vein segments were perfusedsequentially with DC Solution 1, DC Solution 2, and DC Solution 3, witha washing step using sterile water between different DC Solutions. Inthis example, 24 hours Triton X, 8 h TNBP and 16 h DNase was used. Thevein segments were then thoroughly washed with DC Base Solution and PBSfor 1 hour and a 48-hour final wash. Next, the vein segments weresterilized in Sterilization Solution, and were thoroughly washed in PBS(1-hour sterilization washes and 48 hour final wash). Each sterile DCvein segment was then transferred to a container with a permanentlyattached label and frozen at −80° C. in PBS. The compositions of thesolutions used in decellularization and sterilization were provided inTable 1.

TABLE 1 Solutions for decellularization and sterilization SolutionComposition DC Base Sterile water, 5 mM EDTA Solution DC Solution 1 1%Triton X-100 in DC Base Solution DC Solution 2 1% Tri-n-butyl phosphate(TNBP) in DC Base Solution DC Solution 3 40 U/mL DNase I in PBS withCaCl₂ and MgCl₂ Sterilization 0.1% peracetic acid solution in PBSSolution

The acellularity of the decellularized vein scaffolds was verified byhistology with Hematoxylin and Eosin (H&E) staining and4′,6-diamidino-2-phenylindole (DAPI) staining. Specifically, the bluedots in the H&E staining, representing nuclei, were observed in theunprocessed veins but not in the decellularized samples. Similarly,fluorescence from DAPI, indicating DNA, was detectable in theunprocessed veins but not in the decellularized samples. The DNA contentof the decellularized vein segments was also evaluated using a Qubit™fluorometer. The average double-stranded DNA (dsDNA) content of thesamples before decellularization was 152 ng per mg of tissue, with astandard error of the mean (SEM) of 32 ng/mg. After decellularization,the samples contained 0.5 ng dsDNA per mg of tissue, with an SEM of 0.04ng/mg, which was about 0.3±0.03% of the unprocessed material. Therefore,the decellularization rendered the vein scaffolds substantiallyDNA-free.

To recondition/recellularize the vein scaffolds, peripheral whole blood(PWB) samples were taken from recipient pigs by routine venipuncture insterile 10 mL BD vacutainer glass tubes containing 17 IU/mL sodiumheparin. The PWB was mixed with an ex vivo organ perfusion solution(STEEN™ solution) in a 1:1 ratio, and supplemented with 10 ng/mL ofrecombinant human FGF-2 (rhFGF-2), 80 ng/mL of recombinant human VEGF(rhVEGF), and 5 μg/mL of acetylsalicylic acid, thereby generating anautologous blood suspension. The reconditioning/recellularization wasperformed in a closed bioreactor. After prewashing with PBS andpretreatment with heparin, the vein scaffolds were continuously perfusedwith the blood suspension in closed recirculation for 7 days. During theperfusion, the glucose level in the blood suspension was maintainedbetween 3-11 mmol/L. Under these conditions, the cellular and othercomponents from the autologous blood suspension repopulated the veinscaffolds.

Surgery

The reconditioned/recellularized P-TEVs were implanted into therecipient pigs by surgery. Specifically, an incision was made throughlinea alba. A conventional technique to localize vena cava was used forthe first two pigs: the intestines were held aside with gauze wetted insaline and surgery hooks, and the part of vena cava between vena renalisand the bifurcation to vena femoralis was dissected free fromsurrounding tissue. Due to intestinal adhesion formation in one of thesetwo animals, an improved, retroperitoneal technique was used to localizevena cava in the other six pigs (four implanted with P-TEV, twosubjected to sham surgery): the peritoneum and the abdominal wall wereseparated down to vena cava on the dexter side, thereby leaving theintestines untouched. For the P-TEV transplanted pigs, vena cava wascut, and a P-TEV of approximately 4 cm was attached with end-to-endanastomoses. In the sham operated pigs, the tension of the vein did notallow cutting and suturing at two places as with the P-TEV transplants.Instead, vena cava was cut and sutured with one anastomosis.

During the surgery, the pigs were sedated with tiletamine, zolazepam,and medetomidine, and then intubated for anesthesia with isoflurane.Buprenorfin was given during the surgery for post-surgery pain relief.To prevent coagulation and thrombosis, all the pigs were treatedperorally with 160 mg acetylsalicylic acid once daily for one week priorto the surgery, and with 2 mg/kg rivaroxaban twice daily from one daypre-surgery until euthanization. Additionally, 10,000 IU heparin wasadministered intravenously during the surgery.

Conditions of the Pigs After Surgery

Two pigs developed intestinal adhesion after the surgery, and had to beeuthanized prior to the planned end point due to ileus symptoms. One wasa P-TEV pig in which the vena cava was localized with the conventionaltechnique. It had to be euthanized 16 days post-surgery, and theintestines showed massive adhesions at dissection. The other was a shampig euthanized 7 days post-surgery. The peritoneum of this pig brokeduring the surgery, and the intestines had to be handled with wettedgauze and hooks as with the conventional technique.

No intestinal adhesion was observed during dissections of the other sixpigs. Among these six animals, one sham pig and three P-TEV pigs wereeuthanized at the planned end point of 4-5 weeks post-surgery. Two P-TEVpigs had to be euthanized earlier due to complications not related tothe vena cava transplantation. One was euthanized 3 days post-surgery,because the sutures of the abdominal wall broke. The other waseuthanized 17 days post-surgery because of a knee fracture on the leftfront leg.

Characterization of the Transplanted P-TEVs

Angiography was performed under anesthesia prior to euthanization of theone sham pig and three P-TEV pigs that were alive 4-5 weekspost-surgery. Contrast fluid was injected into a femoral vein, and thevena cava was monitored live using a C-bow X-ray. As shown in FIGS.1A-1B, the P-TEV (FIG. 1B) and the sham (FIG. 1A) operated vein wereboth open with free blood flow.

Following euthanization of each pig, the vena cava was examined. The twosham operated veins and six P-TEVs were all open with free blood flowwithout sign of clotting or thrombosis. No blood clot or thrombosis wasobserved at macroscopic examination.

The veins were then sectioned and prepared for H&E staining and DAPIstaining. As shown in FIGS. 2A-2B, in the P-TEV (FIG. 2B) pig euthanized3 days post-surgery, cells were observed in the P-TEV graft by H&E andDAPI staining. The 3-day sample was analyzed as described. Samples afterRC were routinely analyzed as well (these represented the last stage ofunimplanted P-TEV samples). The P-TEV graft was well cellularized in thepig euthanized 17 days post-surgery. Four to Five weeks after thesurgery, the number of cells in the P-TEV (FIG. 2B) appeared to be equalto the native tissue (FIG. 2A). Importantly, intimal hyperplasia, amajor potential complication in this study, was not observed in theimplanted P-TEV.

The vein samples were also characterized by immunohistochemistry. In thepig euthanized two weeks post-surgery, the proximal, center, and distalparts of the P-TEV had a substantial number of cells, and CD31-positivecells could be identified (FIGS. 3A-3D). FIG. 3A shows native vena cavaproximal to the anastomoses. FIGS. 3B-3D show proximal (FIG. 3B), center(FIG. 3C), and distal (FIG. 3D) parts of the P-TEV. Arrow heads indicateCD31-positive cells.

In a pig euthanized 4-5 weeks post-surgery, the proximal, center anddistal parts of the P-TEV had a similar cellular density as the nativevena cava, and the P-TEV lumen was covered with a CD31-positiveendothelium layer (FIGS. 4A-4D). FIGS. 4A-4D are a series ofimmunohistograms showing DAPI staining and CD31 immunostaining of venacava four weeks post-surgery. FIG. 4A shows native vena cava proximal tothe anastomoses. FIGS. 4B-4D show proximal (FIG. 4B), center (FIG. 4C),and distal (FIG. 4D) parts of the P-TEV. Arrow heads indicateCD31-positive cells.

The morphology of endothelial-like cells lining the luminal surface wasvirtually indistinguishable from that of the native vena cava (FIGS.5A-5B). FIGS. 5A-5B is a series of images at 40× magnification showingH&E staining of native vena cava (FIG. 5A) and P-TEV transplant (FIG.5B) four weeks post-surgery. Arrows indicate endothelial cells in thenative tissue and cells with plated endothelial cell-like morphology inthe P-TEV transplant.

In summary, this in vivo study suggested that the P-TEV were safe andfeasible for vein transplantation.

Example 2: Preparation of Physiological Perfusion Solution

900 ml of Dulbeccos Phosphate-Buffered Saline (DPBS) with calcium andmagnesium was continuously stirred at 60 rpm at RT. 74 g of Human SerumAlbumin were added slowly by layering the powder on the surface of theliquid (to avoid clumping) and stirred until completely dissolved.Foaming was avoided by temporarily reducing the rpm. Subsequently 6.7 gof Dextran-40 were added to the solution and stirred until completelydissolved. The pH was titrated to 7.4 using NaOH and the final volumewas adjusted to 1000 ml using DPBS with calcium and magnesium. Thephysiological perfusion solution is then sterile filtered using a lowprotein-binding sterile filter, aliquoted into 25 ml aliquots in sterile50 ml tubes and stored at 2-8° C. for up to 12 months.

Example 3: Preparation of an Alternative Physiological PerfusionSolution (Not Requiring Expensive HSA)

25 ml of sterile blood plasma from the patient were placed in a sterile50 ml tube. 1.5 ml of a 100 g/L sterile-filtered Dextran-40 stocksolution were then added to the plasma and the solution was agitatedcarefully until completely mixed. The sterile physiological perfusionsolution was stored at 2-8° C. for up to 7 days.

Example 4: Additional Variations of Prepared Physiological PerfusionSolution

DPBS with calcium and magnesium containing 70 g/L HSA, 5 g/L Dextran-40and 11 mmol/L Glucose

DPBS with calcium and magnesium containing 40 g/L HSA and 10 g/LDextran-40

Human blood plasma containing additional 30 g/L HSA and 5 g/L Dextran-40

Human blood plasma containing additional 5 g/L Dextran-40 and in total11 mmol/L Glucose

DPBS with calcium and magnesium containing 70 g/L HSA, 5 g/L Dextran-60and 11 mmol/L Glucose

Example 5: Compositions of Commercially Available PhysiologicalPerfusion Solutions

University of Solution Euro Coolins Wisconsin Celsior Perfadex ColloidComponent Glucose Lactobionate, Lactobionate, Dextran Raffinose,Mannitol Hydroxyethyl starch Buffer Phosphates, Phosphates HistidinePhosphates Bicarbonates Antioxidant Allopurinol, Glutathione,Glutathione Mannitol Osmolarity (mOsm/L) 375 330 320 292 Glucose(mmol/L) 180 5 Na⁺ (mmol/L) 10 25 100 138 K+ (mmol/L) 115 120 15 6 Ca²⁺(mmol/L) 0.25 Mg²⁺ (mmol/L) 5 13 0.8 Cl⁻ (mmol/L) 15 20 142

Example 6: Preparation of a Personalized Blood Vessel from an AcellularTubular Scaffold Prepared by Bioprinting

Example 1 describes a method of preparing a personalized blood vesselusing a decellularized tubular scaffold. Alternatively, a personalizedblood vessel is prepared from a bioprinted tubular acellular scaffold.Briefly, the bioprinted blood vessel scaffold is prepared on a polymer(natural or synthetic), which is a gel form, sponge form, foam form,patch form, or a semi-liquid/fluid form. In this method, a polymerscaffold is perfused with whole blood or whole blood diluted in asolution, e.g., a suspension, which includes whole blood, forpreparation of a personalized blood vessel.

Example 7: Implantation of Personalized Blood Vessel

A subject in need of a transplanted blood vessel is selected, and apersonalized blood vessel prepared by the method provided in Example 1or 2 of the present disclosure is implanted/transplanted into thesubject. The subject's prognosis and recovery post-transplantation ismonitored.

An personalized blood vessel disclosed herein is used for treatment ofvarious blood vessel diseases and disorders, such as deep veinthrombosis (DVT), chronic venous insufficiency (CVI), varicose veins,venous ulceration (e.g., venous leg ulceration), and recurrent legcancer (e.g., caused by deep venous reflux and/or venous hypertension).

Specifically, a subject having a blood vessel disease or disorder isselected. The personalized blood vessel is introduced to the subject bysurgery: a segment of native blood vessel afflicted with the disease ordisorder is removed; the personalized blood vessel or a functionalsegment thereof is anastomosed to the native blood vessel, replacing thesegment of the native blood vessel that is removed. The hybrid bloodvessel generated from the surgery is functionally similar or equivalentas a native blood vessel, and ameliorates the symptoms including dullaching, heaviness, or cramping in legs, itching and tingling, pain thatgets worse when standing, pain that gets better when legs are raised,swelling of the legs, redness of the legs and ankles, skin color changesaround the ankles, varicose veins on the surface (superficial),thickening and hardening of the skin on the legs and ankles(lipodermatosclerosis), ulcers on the legs and ankles, and wound that isslow to heal on the legs or ankles.

Example 8

The recellularization/reconditioning process of an acellular tubularscaffold for preparing a personalized blood vessel involved a smallsample of whole blood from the patient. In this example, the process ofrecellularization/reconditioning involving cellular as well asnon-cellular components (e.g., thrombocytes, nucleated cells, proteins,growth factors, signaling factors, immunoglobulins) of the whole bloodare selected or enriched for use in the process. In one example,cellular as well as non-cellular components of the whole blood arecombined with whole blood. Alternatively, the cellular as well asnon-cellular components of the whole blood are used instead of the wholeblood in the recellularization/reconditioning process. The latterprocess is undertaken for process-related reasons, and/or to optimizethe personalization of a blood vessel. Enrichment or selection isperformed using, e.g., centrifugation, gradient centrifugation,selective adhesion, chromatography, filtration, or sorting (FACS, MACS).

Other Embodiments

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. Any definitions provided herein are included for thepurpose of understanding the present subject matter and for constructingthe appended patent claims. Abbreviations used herein have theirconventional meaning within the chemical and biological arts.

What is claimed is:
 1. A method of preparing a personalized blood vesselcomprising, contacting a surface of an acellular tubular scaffold withan undiluted whole blood sample from a subject in need of thepersonalized blood vessel, wherein the contacting is performed for 3days to 21 days.
 2. The method of claim 1, wherein a population of cellsin the whole blood sample populate the acellular tubular scaffold. 3.The method of claim 1, wherein the whole blood sample comprises one ormore non-cellular factors, wherein one or more non-cellular factors ofthe whole blood populate the scaffold, and wherein the non-cellularfactors promote cellularization of the acellular tubular scaffold andhost compatibility of the vessel upon grafting.
 4. The method of claim1, wherein the undiluted whole blood sample further comprises ananti-thrombotic factor.
 5. The method of claim 4, wherein theanti-thrombotic factor comprises an anticoagulant agent.
 6. The methodof claim 5, wherein the anticoagulant agent comprises heparin or adextran.
 7. The method of claim 6, wherein the heparin is present in theundiluted whole blood sample at a concentration from about 0.5 IU/mL toabout 150 IU/mL at the beginning of contacting the surface of theacellular tubular scaffold.
 8. The method of claim 7, wherein theheparin is present in the undiluted whole blood sample at aconcentration of about 6.7 IU/mL at the beginning of contacting thesurface of the acellular tubular scaffold.
 9. The method of claim 6,wherein the dextran is dextran-40.
 10. The method of claim 6, whereinthe dextran is present in the undiluted whole blood sample at aconcentration from about 1 g/L to about 55 g/L at the beginning ofcontacting the surface of the acellular tubular scaffold.
 11. The methodof claim 4, wherein the anti-thrombotic agent comprises ascorbic acid.12. The method of claim 11, wherein the ascorbic acid is present in theundiluted whole blood sample at a concentration from about 0.2 μg/mL toabout 200 μg/mL at the beginning of contacting the surface of theacellular tubular scaffold.
 13. The method of claim 11, wherein theconcentration of ascorbic acid is present in the undiluted whole bloodsample at a concentration of about 5 μg/mL at the beginning ofcontacting the surface of the acellular tubular scaffold.
 14. The methodof claim 4, wherein the anti-thrombotic factor comprises acetylsalicylicacid.
 15. The method of claim 14, wherein the acetylsalicylic acid ispresent in the undiluted whole blood sample at a concentration of fromabout 0.2 μg/mL to about 200 μg/mL at the beginning of contacting thesurface of the acellular tubular scaffold.
 16. The method of claim 14,wherein the acetylsalicylic acid is present in the undiluted whole bloodsample at a concentration of about 5 μg/mL at the beginning ofcontacting the surface of the acellular tubular scaffold.
 17. The methodof claim 1, wherein the whole blood sample further comprises equal to ormore than the population average physiological level of a growth factorselected from the group consisting of: granulocyte macrophage-colonystimulating factor (GM-CSF), interleukin (IL)-3, IL-4, neutrophin(NT)-6, pleiotrophin (HB-GAM), midkine (MK), interferon inducibleprotein-10 (IP-10), platelet factor (PF)-4, monocyte chemotacticprotein-1 (MCP-1), RANTES (CCL-5, chemokine (C-C motif) ligand 5), IL-8,IGFs, fibroblast growth factor (FGF)-1, FGF-2, FGF-3, FGF-4, FGF-5,FGF-6, FGF-7, FGF-8, FGF-9, transforming growth factor (TGF)-13, VEGF,platelet-derived growth factor (PDGF)-A, PDGF-B, HB-EGF, hepatocytegrowth factor (HGF), tumor necrosis factor (TNF)-a, insulin-like growthfactor (IGF)-1, and any combination(s) thereof.
 18. The method of claim17, wherein the growth factor is a fibroblast growth factor (FGF)-2. 19.The method of claim 1, wherein the contacting is performed for 4 days to21 days.
 20. The method of claim 1, wherein the contacting is performedfor 5 days to 21 days.
 21. The method of claim 1, wherein the contactingis performed for 6 days to 21 days.
 22. The method of claim 1, whereinthe contacting is performed for 7 days to 21 days.
 23. The method ofclaim 1, wherein the contacting is performed for 7 days to 9 days. 24.The method of claim 1, wherein the contacting is performed for 3 days to14 days.